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https://f1000research.com/articles/12-127/v1
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02 Feb 23
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{
"type": "Review",
"title": "Monkeypox (mpox) in immunosuppressed patients",
"authors": [
"Sirwan Khalid Ahmed",
"Mona Gamal Mohamed",
"Eman Abdelaziz Dabou",
"Israa Abuijlan",
"Deepak Chandran",
"Nahed A. El-Shall",
"Hitesh Chopra",
"Kuldeep Dhama",
"Mona Gamal Mohamed",
"Eman Abdelaziz Dabou",
"Israa Abuijlan",
"Deepak Chandran",
"Nahed A. El-Shall",
"Hitesh Chopra"
],
"abstract": "The World Health Assembly declared that smallpox had been completely eradicated from the human population in 1980. Monkeypox, a zoonosis native to damp forested regions in West and Central Africa, is the illness that is most comparable to smallpox clinically and immunologically. Both illnesses could be prevented by the smallpox vaccine. Although the monkeypox virus is a less effective human disease than the smallpox virus, it could now spread among human populations if smallpox had not been eradicated and population-wide immunity had not been developed. A health warning on severe monkeypox in people who are immunocompromised due to Human Immunodeficiency virus (HIV) and other illnesses was released by the U.S. Centers for Disease Control and Prevention (CDC) on September 29, 2022. The advise does not specifically include primary immunodeficiency, but it does define other immunocompromising disorders as âhaving autoimmune disease with immunodeficiency as a clinical componentâ. The documented severe signs of monkeypox include widespread rashes with secondary fungal or bacterial skin infections or tissue death (necrosis), intestine obstruction, and difficulties with the heart, lungs, urinary system, and nervous system. Both those with healthy immune systems and those with weakened immune systems, such as those who are immunosuppressed, older people, children, etc., have encountered serious health issues, but the latter group is more likely to do so. According to the advisory, âof the people with severe monkeypox manifestations for whom CDC has been consulted, the majority have had HIV with CD4 counts 200 cells/ml, indicating substantial immunosuppressionâ. The current article goes into great detail about monkeypox disease occurring in immunocompromised patients and preventive guidelines.",
"keywords": [
"monkeypox",
"immunocompromised patients",
"autoimmune disease",
"human immunodeficiency virus",
"management",
"prevention",
"control"
],
"content": "Introduction\n\nThe monkeypox virus (MPXV) is an Orthopoxvirus that can spread to humans and result in monkeypox (mpox) disease quite similar to the smallpox.1 Large respiratory droplets, inadvertent or intentional contact with bodily fluids or lesion material or contact with biotaâincluding bedding or towelsâare some of the main ways that viruses are spread. Symptoms like malaise, headache, lymphadenopathy, myalgia, and cutaneous symptoms have been documented within one to three days of the onset of fever. Macules may initially be present when a lesion first develops, followed by papules, pustules, and vesicles, which then dry up and peel off. Immunocompromised individuals should be examined for other diseases, such as primary or secondary infection with the varicella zoster virus or other pathogens such as Cryptococcus neoformans, Histoplasma capsulatum, or Bartonella henselae, when skin lesions arise along with viral prodromal symptoms.2 The epidemiological history of the mpox revealed that the first recognition of this disease in human beings was in 1970 in the Democratic Republic of the Congo among children.3 The mpox disease was endemic to central and western Africa region where 12 countries were identified, including Benin, Cameroon, the Central African Republic, the Democratic Republic of the Congo, Gabon, the Ivory Coast, Liberia, Nigeria, Sierra Leone, and South Sudan.4â7 Since then, human cases of mpox have occurred rarely in that area.3,8â14 The first outbreak of the disease reported outside Africa was in the USA in 2003.15 Investigation revealed that the USA outbreak was linked to an infected Prairie dog with MPXV. These pets were housed near rodents shipped from Ghana in 2003.16 After the 2003 USA outbreak, several countries reported mpox cases with a history of travel from Nigeria in 2018,17 including Israel,18 the UK,19 and Singapore.20 Now, some 34 years later, we find ourselves in an eerily similar predicament.21\n\nSince several Orthopoxvirus species share genetic and antigenic characteristics, getting infected with one of them may provide significant protection from getting infected with the others. The vaccinia virus vaccine offers defense against illnesses brought on by the variola major, mpox, or cowpox viruses.22,23 Vaccine-induced cross-protection appears to be mediated by a variety of immunologic pathways, with neutralizing antibodies among the key players.24â26 Monkeys can be immunized with the human smallpox vaccine to prevent mpox, which is consistent with the ability of the smallpox vaccine to give cross-protection for humans against mpox. Since the end of smallpox vaccinations in 1978, cross-protective immunity to different orthopoxviruses has decreased, especially in younger people without vaccinia-induced immunity, and the number of unvaccinated, susceptible people has increased globally. In fact, during the past few years, these changes have been accompanied by a rise in the number and geographic dispersion of human mpox cases.27,28 A health warning on severe mpox in people who are immunocompromised due to HIV and other illnesses was released by the U.S. Centers for Disease Control and Prevention (CDC) on 29th September 2022.29 Although âhaving autoimmune disease with immunodeficiency as a clinical componentâ is included in the advisory's definition of other immunocompromising disorders, primary immunodeficiency is not specifically included in it. There have been reports of widespread rashes, subsequent bacterial or fungal skin infections, tissue death (necrosis), bowel obstruction, heart, lung, urinary, and neurological problems as severe symptoms of mpox.25 Serious problems have happened in both immunocompromised and immunocompetent individuals, although immunocompromised individuals are more prone to experience them. The majority of those with severe mpox presentations for whom the CDC has been contacted have Human Immunodeficieny Virus (HIV) and CD4 counts below 200 cells/ml, indicating significant immunosuppression, according to the advice.30\n\nIn-depth discussion of mpox disease in immunocompromised people and recommended preventative measures is provided in the current article.\n\n\nThe ongoing 2022 mpox outbreak\n\nIn the 2022 outbreak, the mpox disease was reported by numerous non-endemic countries. The first case of mpox was reported by the United Kingdom Health Security Agency (UKHSA) in the United Kingdom. Afterwards, cases of the illness were reported in Spain, Portugal, Italy, the United Kingdom, and the United States.31â40 Up to January 06, 2023, the Centers for Disease Control and Prevention (CDC) confirmed 84,075 mpox cases in 110 countries worldwide with 75 deaths,41 and clinical research has shown that the disease has distinct epidemiological and clinical features.31,32,42,43 Statistics show that one-third of the reported cases were highly concentrated in the USA (the highest), Spain, Germany, and the UK.44 The mortality rate of this disease is less severe as investigation revealed that the MPXV genome involved in the current outbreak belongs to West African clade II, which is less virulent than the Central African clade also known as the Congo Basin clade.45 The combined case fatality rate of the West African and Congo Basin MPXV clades is around 8.7% worldwide.46,47\n\nThe demographic characteristics of reported cases distinguished from the previous outbreak were 99% male with a median age of 37 years, and 44% were HIV-positive.48 Furthermore, 99% of reported cases were gay, bisexual, and having men-to-men sex,48 indicating sexual transmission as a new mode of transmission.27 Interestingly, Pluart et al. declared that mpox occurred among healthcare workers.49 However, the protection of healthcare workers shoud be a top prority during this public health emergency.50,51 Scientists have espoused that the affected people represent the population who did not receive smallpox vaccination due to disease eradication, indicating its possible contribution to their susceptibility to mpox disease.52,53\n\nGenital lesions have been reported as one of the distinguishing features of the current outbreak.54 The majority of confirmed cases have reported to clinics with at least one symptom, including systemic rashes, fever, or genital rashes, which is not consistent with the typical presentation of mpox disease, which usually includecentrifugal rashes preceded by fever and swollen lymph nodes.37,46 In addition, the number of rashes is less in most cases, with the first rash occurring in the genital and perineal area followed by monographic and centrifugal distribution all over the body.23,55 The other sites of lesions were as follows: 55% on the truck, arms, and legs, 25% on the face, 10% on the palms and soles.43 Most rashes (58%) were identified as vesiculopustular.43\n\n\nTransmission and replication\n\nThe MPXV is considered one of the largest DNA viruses56 and its size ranges from 130 to 360 kbp.57 It therefore stimulates the host immune system rapidly. The MPXV has two distinctive sets of modulatory proteins responsible for invading the immune system, including intracellular modulatory proteins and extracellular modulatory proteins. Another unique feature of MPXV is its exclusive replication in the cytoplasm.57 The replication process of MPXV starts with viral attachment initiated by glycosaminoglycans including chondroitin, heparin sulfates, and laminin,58 followed by viral fusion to the host cell involving 11 to 12 non-glycosylated, transmembrane proteins.59 Once the viral entry is complete, viral transcription takes place by virus-encoded multi-subunit DNA-dependent RNA polymerase.60 It is proceeded by the translation of proteins on the host ribosome, which occurs at three levels: early, intermediate and late translation.60 Studies reveal that DNA synthesis and replication occur at cytoplasmic structures called factories,18 proving its effectiveness in viral RNA synthesis.56 Each factory derives from the cell's rough endoplasmic reticulum (RER).61 Lastly, some of the mature virions transported via microtubules and coated by endoplasmic reticulum or Golgi-derived membranes will exit the cell by fusing with the cytoplasmic membrane and become extracellular enveloped virus (EEV).62\n\nThe orthopoxviral physical stability contributes to their varied modes of transmission.45 The mpox information chain includes animal-to-human transmission and human-to-human transmission. Since the virus is present in the infected host lesions, crust, and secretions, direct contact with these secretions can promote viral transmission, whether the host is animal or human. Animal to human transmission occurs when there is direct contact with the bodily fluids of affected animals, such as blood, saliva, and cutaneous and mucosal lesions of these animals. Furthermore, respiratory droplets and eating raw meat or poorly cooked meat products of affected animals can transmit the MPXV. Another transmission mode includes the bites and scratches caused by infected animals.63 Monkeys, rats, and squirrels are the primary host of MPXV in Africa.51\n\nSimilarly, transmission from human to human occurs when there is direct contact with an infected person's rashes, sores, scabs, respiratory droplets, or oral fluids.44,46 Furthermore, sharing the same household of infected individuals increases the risk of contamination.63 The common mechanism used by MPXV in the 2022 outbreak is attacking the host defense by encoded proteins produced during transcriptions named MHC class II antigen presentation inhibitor, an IFN-alpha/beta receptor glycoprotein and IL-1/TLR signaling inhibitor.64 Moreover, vertical transmission, such as from mother to fetus, was also identified.65 Another unique feature that contributed to the current outbreak is the excessive genome mutations that result from the action of apolipoprotein B mRNA-editing catalytic polypeptide-like 3(APOBEC3) enzymes.64 The action of these enzymes results in hypermutated, viable variants of the virus as revealed by phylogenomic analysis.64\n\nIn the current outbreak, there is a disappearance of an epidemiological link to the endemic region.27 Yet, data showed that 91% of confirmed cases reported an immediate sexual exposure before symptoms started, particularly with men having sex with men,23 while 98% of the patients identified as gay or bisexual.43 Therefore, WHO considered sexual exposure in the 21 days before symptoms onset as a risk factor,44,46 especially among men who have sex with other men. Evidence showed that all confirmed cases of mpox among men who have sex with other men have a history of sexually transmitted disease (STD), hepatitis C, syphilis, and HIV.33 Epidemiologists reported that the reproductive ratio value of MPXV is between 1.10 and 2.40, suggesting the ability of an infected individual to infect one to two other persons.66 Such value highlights the need to initiate preventive measures to contain the spread of the disease.50,63,67\n\n\nImmunosuppression\n\nThe immune system plays a vital role in defending the body against microorganisms and other foreign bodies.68 Failure of the immune system to protect the body indicates the body's immunosuppressed status. Immunosuppression is defined as âa state of temporary or permanent dysfunction of the immune response resulting from insults to the immune system and leading to increased susceptibility to diseaseâ, originally proposed by Dohms and Saif in 1984.69,70 Immune dysfunction is classified as primary and secondary immune dysfunction. The immunosuppression status occurs due to disease condition or medication-induced immunosuppressed state. Consequently, hematologic malignancies, solid-organ transplant, chimeric antigen receptor (CAR)-T-cell therapy, or hematopoietic stem cell transplant are all common causes of immunosuppression, as are moderate to severe levels of immunodeficiency disease, such as DiGeorge syndrome, Wiskott-Aldrich syndrome, HIV cases with CD4 cell counts less than 200/mm3, and the history of an AIDS-defining illness without immune recrudescence. The degree of immunosuppression can vary among patients.71 Furthermore, medication that induces immunosuppression status includes high-dose corticosteroids (i.e., â¥20 mg of prednisone for two or more weeks), alkylating agents, antimetabolites, transplant-related immunosuppressive drugs, tumor necrosis factor (TNF) blockers, and other biologic agents that are immunosuppressive or immunomodulatory. Evidence has shown a steady rise in the number of acquired immunity deficiency (secondary immunodeficiency) conditions in response to an increasing number of individuals with transplantation of solid organ or hematopoietic stem cells72 as such medical treatment requires an intensive immunosuppressive regimen which can cause the patient to develop severe adenovirus infections.72 A US cross-sectional study revealed that 2.8% of patients experienced drug-induced immunosuppression between 2018 and 2019, particularly those who were prescribed oral corticosteroids for 30 days or longer (40.9%).73 Oral corticosteroids, methotrexate, and other disease-modifying anti-rheumatic therapies, transplant antirejection drugs, tumor necrosis factor inhibitors, antineoplastic treatments, and other biological product medications all contribute to immunosuppression.73 Immunosuppressive medication has a significant role in patient survival such as keeping a person from rejecting an organ transplant and treating the overactivity of the immune system in cases like autoimmune disease and allergies. On the other hand, immunosuppressant medication can negatively impact a patients' health as it increases their likelihood of having infections.74\n\nThe most commonly reported infection among immunocompromised patients is a protracted infection characterized by the intra-host viral revolution and the generation of multiply mutated viruses.74 The common form of protracted infection is protracted bacterial bronchitis which occurs among children.75 In addition, other types of infections associated with immunosuppression are viral, bacterial, and fungal infections of the blood, lungs, and central nervous system.76 Furthermore, the immunocompromised status might increase patients' susceptibility to prolonged infection, resulting in a prolonged length of stay in hospital and more complications.77 Studies on immunosuppressed patients' response to infection vary in terms of mortality rate, complication, and length of stay. For example, authors of a study conducted in Spain among immunocompromised patients admitted with Influenza A (H1N1) virus in 2009 reported that immunocompromised patients had higher mortality than non-immunosuppressed individuals.78 In addition, the complicated cases experienced a bacterial coinfection, specifically gram-negative bacilli and Staphylococcus aureus infections.78 Inversely, a retrospective cohort study in the U.S. in 2020 was conducted on the impact of COVID-19 on drug-induced immunosuppressed patients. It revealed that the chronic use of immunosuppressive drugs was neither associated with worse nor better clinical outcomes among hospitalized cases with COVID-19 in terms of the risk of using mechanical ventilation, in-hospital mortality, or length of stay.79 Similarly, another study revealed that immunosuppressed patients infected with COVID-19 are not at increased risk of severe pulmonary disease compared to other populations, highlighting the necessity of continuing patients' treatment, such as chemotherapy and radiotherapy.80 The controversial condition of immune suppression was pregnancy. There was a debate regarding pregnancy being considered an immunosuppression state.81 However, the authors highlight that the susceptibility of pregnant women to infection depends on the placental immune response to certain pathogens.81,82\n\n\nMonkeypox in immunosuppressed patients\n\nAccording to studies, there is a greater chance of severe manifestation of MPXV infection in immunosuppressed patients.42,83,92,84â91 Immunosuppressed patients include patients who have undertaken cancer treatment, organ transplant, HIV infection, primary immune deficiency disorders, some severe autoimmune disorders, and medications to treat autoimmune diseases and other illnesses that can weaken the immune system. The disease appears to have a lethal prognosis, especially in children who have not been vaccinated against smallpox.83 Individuals with compromised immune systems include those with HIV, hematological malignancies, usage of immunosuppressive medicines such corticosteroids, organ transplant patients, autoimmune illness, or innate immunodeficiencies.\n\nPatients with immune impairment run the risk of contracting serious illness. As a result, efforts should be undertaken to strengthen their immune system in addition to using tecovirimat (e.g., limiting the use of immunosuppressive medications, initiating antiretroviral therapy for those with HIV). Following a tecovirimat 14-day therapy, certain individuals with significant immunocompromise may continue to develop lesions.93 If viral resistance is suspected in this situation, physicians may think about sending a second swab for sequencing. As long as there is no sign of viral resistance, it is reasonable to continue tecovirimat (with emphasis on the need for a fatty meal for optimal absorption) until there is clinical improvement, but no longer than 90 days.94 In contrast, if there is sign of viral resistance, a second anti-viral medication, such as cidofovir (or brincidofovir, if it becomes available), can be added. An infectious disease expert or public health official should be consulted for the management of such patients (e.g., the CDC mpox consultation team in the United States.26,29,67,95\n\nThe immunocompromised patient is more liable to get the mpox infection with severe manifestations of mpox, including extensive skin rashes with secondary bacterial or fungal infections or necrosis, bowel blockage, and heart, lung, urinary, and neurological complications.34,53,86 Guarner et al., (2022)27 reported that an immunosuppressed patient had several scattered maculopapular rashes and pustules on the trunk, upper and lower extremities, groin, and peri-anal area and palpable cervical lymph nodes. Moreover, immunocompromised patients are more likely to develop complications, including respiratory deterioration, acute kidney injury, and multiple organ dysfunctions.83 Simon-Gozalbo et al. (2022)96 reported a case of a 30-year-old male patient diagnosed with HIV infection. The manifestations were maculopapular rash affecting mainly the trunk, buttocks, upper and lower extremities, and multiple demarcated purpuric macules with central umbilicated pustules and crusts, along with palpable cervical lymph nodes.\n\n\nManagement and prevention\n\nPrevention is essential for the immunocompromised patient to protect them from infection. There are two types of vaccines: second and third generation. Second-generation vaccines called replication-competent vaccinia virus vaccines due to their replication capacity in mammalian cells (examples include ACAM2000 and APSV approved for >18 years old) are associated with complications among immunocompromised patients.97 This type of vaccine is not recommended for any group of immunosuppressed patients, especially those with inflammatory/autoimmune diseases of the central nervous system where there can be an outbreak of the disease. Third-generation vaccines, for example, MVA-BN/JYNNEOS/Imvamune, are highly attenuated live vaccines. In contrast to replication-competent smallpox vaccines, these replication-deficient vaccinia vaccines pose a lower risk of side effects, making them suitable for use in healthy people as well as those with immune system deficits, HIV infection, atopic dermatitis, or allergic rhinitis. However, those with compromised immune systems may not respond as well to the vaccine.98,99 The vaccination of those with immunosuppression, who are at high risk for developing mpox sequelae, should proceed with a 0.5 ml SC/IM dose. People living with HIV whose CD4 count is greater than 200 cells/mm3 can get MVA-BN through intradermal administration.100\n\nJYNNEOS (also known as Imvamune and Imvanex) is a vaccine against a modified Vaccinia Ankara virus that does not replicate. In the US, it was approved for use in preventing both mpox and smallpox.101 Because JYNNEOS does not cause the development of live viruses in vaccinated patients, it is safer for use in immunocompromised individuals than ACAM2000. Importantly, however, immunocompromised persons may have a lower immunological response to the JYNNEOS vaccination. Thus, protection can be weaker than in immunocompetent people. Both vaccines can legally be administered to those aged 18 and up. However, information on JYNNEOS's ability to protect humans from MPXV infection is scant.26,46,102 There is a lack of information regarding the efficacy of vaccinia immune globulin (VIG) in the treatment of mpox sequelae. In severe cases of human mpox, VIG may be considered, albeit its efficacy is uncertain. In the case of an exposed person with significantly compromised cellular immunity, for whom smallpox vaccination is not an option, VIG may be considered as a prophylactic measure.1\n\nIn conclusion, the available vaccines for smallpox today are also approved for mpox due to the 85% effectiveness provided by cross-immunity. However, before recommending or not vaccinating immunosuppressed patients, it is essential to be well informed of the type of vaccine that local authorities are obtaining. If it is a second-generation live attenuated vaccine, these patients should not receive it. If it is a third generation live attenuated vaccine with no replication capacity and proven safe in immunosuppressed patients. In that case, vaccination should be advised, always considering that the underlying disease should not be active at the moment of the inoculation.2,103\n\nNo licensed treatment or proper evidence-based guideline is available for treating human mpox. However, the viruses belonging to the Orthopoxvirus genus are genetically similar. Hence, antivirals used to treat smallpox might be effective against mpox. Antiviral drugs such as tecovirimat, cidofovir, and brincidofovir can be considered mainly for those with severe symptoms or who may be at risk of poor outcomes, such as those with immune suppression.104\n\nData on mpox in HIV patients are sparse; however, early identification, treatment, and prevention may lessen the severity of potential complications and slow the disease's transmission.84 Two anti-viral medications, tecovirimat and brincidofovir, have been given the green light by the Food and Drug Administration (FDA) to treat smallpox. The Centers for Disease Control and Prevention (CDC) offers tecovirimat for use in the treatment of mumps as a first-line agent. Co-administration of tecovirimat and antiretroviral therapy (ART) for HIV infection is safe because there are no known medication interactions that would prevent it. The JYNNEOS vaccination can be used for prophylaxis both before and after exposure if necessary. Although the European Medicines Agency (EMA) has given its approval for the use of tecovirimat in mpox, there is a need for innovative therapies with different mechanisms due to the danger of resistance. In terms of effectiveness and safety, tecovirimat is the drug of choice for treating mpox. Consequently, the accessibility and diversity of effective anti-Orthopoxvirus medicines will be improved through the target-based design of novel antivirals. Patients who did not show a response to tecovirimat run the risk of developing resistance. An immunocompromised HIV/AIDS patient was reported to have contracted mpox by Viguier et al. (2022).88 He had a serious, prolonged infection that was treated with tecovirimat for 14 days after his clinical status deteriorated. Almost immediately, he began to feel better, and both the skin lesions and MPXV burdens dropped with no negative consequences. In this instance, tecovirimat shows promise as an effective treatment. The success of tecovirimat in this patient suggests that it may be used to treat other immunocompromised people who have contracted MPXV. Hernandez et al. (2022)86 reported a case study about an immunocompromised patient and treated him with tecovirimat. Tecovirimat could effectively and safely treat severe mpox infections among immunocompromised patients. Last but not least, there is an immediate need to focus on minimizing psychological distress, particularly among immunocompromised individuals and healthcare workers.95,105,106 An overview of mpox in immunosuppressed patients, preventive measures and clinical management is presented in Figure 1.\n\n\nConclusion and future prospects\n\nImmunotherapeutics and preventative strategies are critical public health interventions that complement rigorous contact tracing in stopping the spread of mpox. In a similar manner, serology-based investigations are effective surveillance tools for tracing contacts and determining exposure histories. However, given that vaccination provides a baseline of protection against poxviruses, these serological diagnostic approaches must be MPXV-specific. Expanding the surveillance network and identifying gaps are also crucial for an efficient ring-fencing system. Importantly, there is a fundamental need in public health to alert individuals who may be exposed about the advantages and hazards of vaccination.\n\nNumerous important scientific queries are yet unanswered. The mechanisms of immune defense against the MPXV will need to be better understood, which will necessitate more research on the human systemic and mucosal immune responses during MPXV infection. It is significant to note that it is not yet known whether previous exposure to variola, mpox, or smallpox vaccine results in any type of mucosal immunity. Understanding the mucosal immune responses will be critical due to the respiratory aerosol transmission of MPXV and other poxviruses. To further understand the respiratory difficulties produced by MPXV, it would be especially necessary to understand the functions of tissue-resident memory T cells and IgA in infection. Because MPXV DNA has been detected in semen, it is also necessary to define the immunity of the preputial mucosa. A key objective for assessing more recent vaccines, particularly those intended for the at-risk populations of immune compromised populations such as older people, pregnant women and children, is defining the immunological correlates of protection. What other factors, such as those related to behavior, geography, nutrition, health, immunology, or genetics, besides not having received a vaccine, could be at play? Recent research suggests that the severity of a viral respiratory infection in young children is tied to the efficiency of their innate immune responses. Children who are infected typically have weaker T cell and B cell responses than adults, similar to reports for SARS-CoV-2 infections. Children with MPXV tend to manifest a more severe disease and have lower vaccine effectiveness, which may be explained by the characterization of adaptive immune responses in these children. Understanding the risks of vaccination in immunocompromised populations, particularly children and expectant women, is also crucial.\n\nIt is feasible for MPXV to co-infect with any of the several infectious diseases that are endemic at global level. A co-infection with malaria and an alphavirus, for instance, can drastically alter host immunity and influence the course of an illness. Co-infection with other illnesses that disproportionately affect the group of men who have sex with men makes it especially important to understand mpox disease and immunization, especially given the continuous transmission among these communities in non-endemic countries. This is especially true for HIV-1, which can significantly inhibit adaptive immune reactions. Additionally, it is important to identify the risk factors for severe MPXV. The most vulnerable groups are known to be unvaccinated people, pregnant women, and young children. Other immunocompromised populations, such as older people, those using long-term drugs, and those with underlying metabolic illnesses that may exhibit the disease in a different way, also need to be characterized though.\n\nPatients, including immunocompromised humans such as geriatric patients or babies born to mothers infected with (MPXV) should be closely monitored for the development of chronic health problems. The Zika virus epidemic provided evidence that children who were exposed to the virus in utero may be at risk of developmental issues later in life.107 Evidence suggests that just 25% of pregnant women infected with MPXV would be successful in giving birth108; there should be a higher priority placed on this area of research. Furthermore, infants and young children appear to be more vulnerable to severe mpox.109,110 Data on fetal development after congenital MPXV infection are currently missing. The ability to determine whether MPXV infection can have long-term repercussions, as was seen after SARS-CoV-2 infection during the current pandemic, would also be made possible by longitudinal surveillance of patients with the MPXV.",
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Publisher Full Text\n\nNg OT, Lee V, Marimuthu K, et al.: A case of imported Monkeypox in Singapore. Lancet Infect. Dis. 2019; 19(11): 1166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSherwat A, Brooks JT, Birnkrant D, et al.: Tecovirimat and the treatment of monkeypoxâpast, present, and future considerations. N. Engl. J. Med. 2022; 387(7): 579â581. PubMed Abstract | Publisher Full Text\n\nBrown K, Leggat PA: Human monkeypox: current state of knowledge and implications for the future. Trop. Med. Infect. Dis. 2016; 1(1): 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBragazzi NL, Kong JD, Mahroum N, et al.: Epidemiological trends and clinical features of the ongoing monkeypox epidemic: A preliminary pooled data analysis and literature review. J. Med. Virol. 2022; 95. Publisher Full Text\n\nNguyen P-Y, Ajisegiri WS, Costantino V, et al.: Reemergence of human monkeypox and declining population Immunity in the context of urbanization, Nigeria, 2017â2020. Emerg. Infect. Dis. 2021; 27(4): 1007â1014. PubMed Abstract | Publisher Full Text\n\nBunge EM, Hoet B, Chen L, et al.: The changing epidemiology of human monkeypoxâA potential threat? A systematic review. PLoS Negl. Trop. Dis. 2022; 16(2): e0010141. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSah R, Humayun M, Baig E, et al.: FDAâs authorized âJYNNEOSâ vaccine for counteracting monkeypox global public health emergency; an updateâCorrespondence. Int. J. Surg. 2022; 107: 106971. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuarner J, Del Rio C, Malani PN: Monkeypox in 2022âWhat Clinicians Need to Know. JAMA 2022; 328: 139â140. PubMed Abstract | Publisher Full Text\n\nHarapan H, Ophinni Y, Megawati D, et al.: Monkeypox: A Comprehensive Review. Viruses 2022; 14(10): 2155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChandran D, Dhama K, Chakraborty S, et al.: Monkeypox: An update on current knowledge and research advances. J. Exp. Biol. Agric. Sci. 2022; 10: 679â688. Publisher Full Text\n\nLum F-M, Torres-Ruesta A, Tay MZ, et al.: Monkeypox: disease epidemiology, host immunity and clinical interventions. Nat. Rev. Immunol. 2022; 22(10): 597â613. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCatalà A, Clavo-Escribano P, Riera-Monroig J, et al.: Monkeypox outbreak in Spain: clinical and epidemiological findings in a prospective cross-sectional study of 185 cases. Br. J. Dermatol. 2022; 187(5): 765â772. PubMed Abstract | Publisher Full Text\n\nTarÃn-Vicente EJ, Alemany A, Agud-Dios M, et al.: Clinical presentation and virological assessment of confirmed human monkeypox virus cases in Spain: a prospective observational cohort study. Lancet 2022; 400(10353): 661â669. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAntinori A, Mazzotta V, Vita S, et al.: Epidemiological, clinical and virological characteristics of four cases of monkeypox support transmission through sexual contact, Italy, May 2022. Eurosurveillance. 2022; 27(22): 2200421. Publisher Full Text\n\nDuque MP, Ribeiro S, Martins JV, et al.: Ongoing monkeypox virus outbreak, Portugal, 29 April to 23 May 2022. Eurosurveillance. 2022; 27(22): 2200424.\n\nCaria J, Pinto R, Leal E, et al.: Clinical and Epidemiological Features of Hospitalized and Ambulatory Patients with Human Monkeypox Infection: A Retrospective Observational Study in Portugal. Infect Dis Rep. 2022; 14(6): 810â823. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdler H, Gould S, Hine P, et al.: Clinical features and management of human monkeypox: a retrospective observational study in the UK. Lancet Infect. Dis. 2022; 22(8): P1153âP1162.\n\nGirometti N, Byrne R, Bracchi M, et al.: Demographic and clinical characteristics of confirmed human monkeypox virus cases in individuals attending a sexual health centre in London, UK: an observational analysis. Lancet Infect. Dis. 2022; 22(9): 1321â1328. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPhilpott D: Epidemiologic and clinical characteristics of monkeypox casesâUnited States, May 17âJuly 22, 2022. MMWR Morb. Mortal. Wkly Rep. 2022; 71: 1018â1022. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiller MJ, Cash-Goldwasser S, Marx GE, et al.: Severe Monkeypox in Hospitalized Patients-United States, August 10-October 10, 2022. MMWR Morb. Mortal. Wkly Rep. 2022; 71(44): 1412â1417. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKraemer MUG, Tegally H, Pigott DM, et al.: Tracking the 2022 monkeypox outbreak with epidemiological data in real-time. Lancet Infect. Dis. 2022; 22: 941â942. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCenters for Disease Control and Prevention (CDC): 2022 Monkeypox Outbreak Global Map.2022 [cited 2022 Oct 5].Reference Source\n\nPatel A, Bilinska J, Tam JCH, et al.: Clinical features and novel presentations of human monkeypox in a central London centre during the 2022 outbreak: descriptive case series. BMJ 2022; 378. Publisher Full Text\n\nThornhill JP, Barkati S, Walmsley S, et al.: Monkeypox virus infection in humans across 16 countriesâAprilâJune 2022. N. Engl. J. Med. 2022; 387: 679â691. PubMed Abstract | Publisher Full Text\n\nSah R, Abdelaal A, Reda A, et al.: Monkeypox and its Possible Sexual Transmission: Where are we now with its evidence? Pathogens. 2022; 11(8): 924. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchnierle BS: Monkeypox Goes North: Ongoing Worldwide Monkeypox Infections in Humans. Viruses 2022; 14(9): 1874. 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Open Forum Infectious Diseases Oxford University Press US; 2022; p. ofac520.\n\nIbrahim PK, Abdulrahman DS, Ali HM, et al.: The 2022 monkeypox outbreakâSpecial attention to nursesâ protection should be a top priority. Ann Med Surg. 2022; 82: 104615. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoyal L, Ajmera K, Pandit R, et al.: Prevention and treatment of monkeypox: a step-by-step guide for healthcare professionals and general population. Cureus. 2022; 14(8). Publisher Full Text\n\nOkyay RA, Bayrak E, Kaya E, et al.: Another Epidemic in the Shadow of Covid 19 Pandemic: A Review of Monkeypox. Proteins 2022; 7: 10. Publisher Full Text\n\nPetersen E, Kantele A, Koopmans M, et al.: Human monkeypox: epidemiologic and clinical characteristics, diagnosis, and prevention. Infect. Dis. Clin. 2019; 33(4): 1027â1043. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith GL, Vanderplasschen A, Law M: The formation and function of extracellular enveloped vaccinia virus. J. Gen. Virol. 2002; 83(12): 2915â2931. Publisher Full Text\n\nKaler J, Hussain A, Flores G, et al.: Monkeypox: A Comprehensive Review of Transmission, Pathogenesis, and Manifestation. Cureus. 2022; 14(7). Publisher Full Text\n\nIsidro J, Borges V, Pinto M, et al.: Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat. Med. 2022; 28(8): 1569â1572. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar N, Acharya A, Gendelman HE, et al.: The 2022 outbreak and the pathobiology of the monkeypox virus. J. Autoimmun. 2022; 131: 102855. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrant R, Nguyen L-BL, Breban R: Modelling human-to-human transmission of monkeypox. Bull. World Health Organ. 2020; 98(9): 638â640. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAhmed SK, Omar RM, Hussein SH, et al.: Middle East countries preparedness for Monkeypox outbreak: A call to action. Int. J. Surg. 2022; 106: 106948. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarshall JS, Warrington R, Watson W, et al.: An introduction to immunology and immunopathology. Allergy, Asthma Clin. Immunol. 2018; 14(2): 1â10.\n\nDohms JE, Saif YM: Guest Editorial: Criteria for Evaluating Immunosuppression. Avian Dis. 1984; 28(2): 305â310. PubMed Abstract | Publisher Full Text\n\nKaspers B, Schat KA, Göbel T, et al.: Avian immunology Academic Press;2021.\n\nLu X, Wang X, Gao Y, et al.: Efficacy and safety of corticosteroids for septic shock in immunocompromised patients: A cohort study from MIMIC. Am. J. Emerg. Med. 2021; 42: 121â126. PubMed Abstract | Publisher Full Text\n\nEchavarrÃa M: Adenoviruses in immunocompromised hosts. Clin. Microbiol. Rev. 2008; 21(4): 704â715. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWallace BI, Kenney B, Malani PN, et al.: Prevalence of immunosuppressive drug use among commercially insured US adults, 2018-2019. JAMA Netw. Open 2021; 4(5): e214920âe214920. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaidar G, Mellors JW: Improving the outcomes of immunocompromised patients with coronavirus disease 2019. Clin. Infect. Dis. 2021; 73(6): e1397âe1401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChang AB, Upham JW, Masters IB, et al.: Protracted bacterial bronchitis: the last decade and the road ahead. Pediatr. Pulmonol. 2016; 51(3): 225â242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoffman JJ, Sullivan NJ: Davis Advantage for Medical-Surgical Nursing: Making Connections to Practice FA Davis;2020.\n\nChoi B, Choudhary MC, Regan J, et al.: Persistence and evolution of SARS-CoV-2 in an immunocompromised host. N. Engl. J. Med. 2020; 383(23): 2291â2293. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCordero E, Aydillo T, Farinas MC, et al.: Immunosuppressed patients with pandemic influenza A 2009 (H1N1) virus infection. Eur. J. Clin. Microbiol. Infect. Dis. 2012; 31(4): 547â556. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndersen KM, Mehta HB, Palamuttam N, et al.: Association between chronic use of immunosuppresive drugs and clinical outcomes from coronavirus disease 2019 (COVID-19) hospitalization: a retrospective cohort study in a large US health system. Clin. Infect. Dis. 2021; 73(11): e4124âe4130. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDâAntiga L: Coronaviruses and immunosuppressed patients: the facts during the third epidemic. Liver Transpl. 2020; 26(6): 832â834. PubMed Abstract | Publisher Full Text\n\nArmenti VT, Moritz MJ, Cardonick EH, et al.: Immunosuppression in pregnancy. Drugs 2002; 62(16): 2361â2375. Publisher Full Text\n\nMor G, Cardenas I: The immune system in pregnancy: a unique complexity. Am. J. Reprod. Immunol. 2010; 63(6): 425â433. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMenezes YR, de Miranda AB : Severe disseminated clinical presentation of monkeypox virus infection in an immunosuppressed patient: first death report in Brazil. Rev. Soc. Bras. Med. Trop. 2022; 55: e0392. Publisher Full Text\n\nOâShea J: Interim guidance for prevention and treatment of monkeypox in persons with HIV infectionâUnited States, August 2022. MMWR Morb. Mortal. Wkly Rep. 2022; 71: 1023â1028. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoffmann C, Jessen H, Wyen C, et al.: Clinical characteristics of monkeypox virus infections among men with and without HIV: a large outbreak cohort in Germany. HIV Med. 2022. PubMed Abstract | Publisher Full Text\n\nHernandez LE, Jadoo A, Kirsner RS: Human monkeypox virus infection in an immunocompromised man: trial with tecovirimat. Lancet 2022; 400(10355): e8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nManoharan A, Braz BX, McBride A, et al.: Severe monkeypox with superimposed bacterial infection in an immunocompetent patient: A case report. IDCases. 2022; 30: e01626. PubMed Abstract | Publisher Full Text | Free Full Text\n\nViguier C, de Kermel T , Boumaza X, et al.: A severe monkeypox infection in a patient with an advanced HIV infection treated with Tecovirimat: clinical and virological outcome. Int. J. Infect. Dis. 2022; 125: 135â137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartÃnez CAP, Flores GAS, SantamarÃa FP, et al.: Monkeypox and its broad clinical spectrum in immunocompromised patients: Two case reports. IDCases. 2022; 31: e01651. Publisher Full Text\n\nde Sousa D , PatrocÃnio J, Frade J, et al.: Human monkeypox coinfection with acute HIV: an exuberant presentation. Int. J. STD AIDS 2022; 33(10): 936â938. PubMed Abstract | Publisher Full Text\n\nWong L, Gonzales-Zamora JA, Beauchamps L, et al.: Clinical presentation of Monkeypox among people living with HIV in South Florida: a case series. Le Infez Med. 2022; 30(4): 610.\n\nRaccagni AR, Candela C, Mileto D, et al.: Breakthrough Monkeypox Infection among Individuals Previously Immunized with Smallpox or Monkeypox Vaccination. J. Infect. 2022; 86: 154â225. Publisher Full Text Reference Source\n\nPeter OJ, Kumar S, Kumari N, et al.: Transmission dynamics of Monkeypox virus: a mathematical modelling approach. Model Earth Syst Environ. 2022; 8(3): 3423â3434. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRanganath N, Tosh PK, OâHoro J, et al.: Monkeypox 2022: Gearing Up for Another Potential Public Health Crisis. Mayo Clin. Proc. 2022; 97(9): 1694â1699. 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Publisher Full Text\n\nGalleguillos L: Monkeypox and Immunosuppressed Neurological Patients: What can we Advise Vaccination in Multiple Sclerosis and Neuromyelitis Optica Patients? J. ISSN. 2022; 3: 1177â1179. Publisher Full Text\n\nUK Health Security Agency: Recommendations for the use of preand post-exposure vaccination during a monkeypox incident.2022 [cited 2022 Dec 12].Reference Source\n\nAhmed SK: Prevention, Vaccination, Management and Infection Control of Monkeypox Outbreak: an Update Global Recommendation for the Next Year 2023. J. Pure Appl. Microbiol. 2022; 16(1): 3189â3191. Publisher Full Text\n\nTitanji BK, Tegomoh B, Nematollahi S, et al.:Monkeypox: A Contemporary Review for Healthcare Professionals. Open Forum Infectious Diseases Oxford University Press; 2022; p. ofac310.\n\nChakraborty S, Mohapatra RK, Chandran D, et al.: Monkeypox vaccines and vaccination strategies: Current knowledge and advances. An updateâCorrespondence. Int. J. Surg. 2022; 105: 106869. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarahat RA, Sah R, El-Sakka AA, et al.: Human monkeypox disease (MPX). Infez. Med. 2022; 30(3): 372â391. PubMed Abstract | Publisher Full Text\n\nAhmed SK, M-Amin HI, Abdulqadir SO, et al.: Timely mental health care for the 2022 novel monkeypox outbreak is urgently needed. Ann Med Surg. 2022; 82: 104579. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAhmed SK, Saied AA, Ravi RK, et al.: The 2022 monkeypox outbreak and associated psychiatric morbidities. Int. J. Surg. 2022; 106: 106913. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAguilar Ticona JP, Nery N Jr, Ladines-Lim JB, et al.: Developmental outcomes in children exposed to Zika virus in utero from a Brazilian urban slum cohort study. PLoS Negl. Trop. Dis. 2021; 15(2): e0009162. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMbala PK, Huggins JW, Riu-Rovira T, et al.: Maternal and fetal outcomes among pregnant women with human monkeypox infection in the Democratic Republic of Congo. J. Infect. Dis. 2017; 216(7): 824â828. PubMed Abstract | Publisher Full Text\n\nLopes Moreira ME, Nielsen-Saines K, Brasil P, et al.: Neurodevelopment in infants exposed to Zika virus in utero. N. Engl. J. Med. 2018 Dec 13; 379(24): 2377â2379. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeçanha PM, Gomes Junior SC, Pone SM, et al.: Neurodevelopment of children exposed intra-uterus by Zika virus: A case series. PloS One. 2020; 15(2): 229434. Publisher Full Text"
}
|
[
{
"id": "162327",
"date": "09 Feb 2023",
"name": "Jawad Basit",
"expertise": [
"Reviewer Expertise Cardiology",
"Novel infections",
"Malignancies and hematology",
"gastroenterology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis literature review beautifully explains the recently discovered monkeypox infection in the context of immunocompromised patients. The article has given authoritative references for different figures and all the data has been comprehensively put forward and has a natural flow and coherence. The figure is well made and is up to mark. However, I would like to recommend to add more details on the underlying mechanisms of pathophysiological details in immunocompromised individuals. Moreover, the current epidemiological status of monkeypox needs to be more stressed in the introduction1,2,3.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "9497",
"date": "03 Apr 2023",
"name": "Sirwan Ahmed",
"role": "Author Response",
"response": "Reviewer: 1 Comments to the Author This literature review beautifully explains the recently discovered monkeypox infection in the context of immunocompromised patients. The article has given authoritative references for different figures and all the data has been comprehensively put forward and has a natural flow and coherence. The figure is well made and is up to mark. Answer: Authors are very thankful to the reviewer for devoting time for improving the quality of the manuscript. The inputs and suggestions from the reviewer have been taken under consideration and have been implemented in the manuscript. However, I would like to recommend to add more details on the underlying mechanisms of pathophysiological details in immunocompromised individuals. Moreover, the current epidemiological status of monkeypox needs to be more stressed in the introduction1,2,3. Answer: The authors have updated the manuscript using the information gathered from following references References 1. Saeed S, Shabbir H, Basit J, Ur Rehman ME: Monkeypox: Potentially another pandemic or a mere hoax?. Ann Med Surg (Lond). 2022; 81: 104364 PubMed Abstract | Publisher Full Text 2. Beer EM, Rao VB: A systematic review of the epidemiology of human monkeypox outbreaks and implications for outbreak strategy.PLoS Negl Trop Dis. 2019; 13 (10): e0007791 PubMed Abstract | Publisher Full Text 3. Bunge EM, Hoet B, Chen L, Lienert F, et al.: The changing epidemiology of human monkeypox-A potential threat? A systematic review.PLoS Negl Trop Dis. 2022; 16 (2): e0010141 PubMed Abstract | Publisher Full Text 4. Jang W, Kandimalla L, Rajan S, Abreu R, Campos JE. Monkeypox in an immunocompromised patient with underlying human immunodeficiency virus and syphilis infections in Southern Florida of the United States: a case report. AIDS Res Ther. 2023 Feb 18;20(1):12. doi: 10.1186/s12981-023-00504-4. PMID: 36800970; PMCID: PMC9937856. 5. Dammann F, Raja M, Camargo JF. Progression of human monkeypox infection despite tecovirimat in an immunocompromised adult. Transpl Infect Dis. 2023 Feb;25(1):e14022. doi: 10.1111/tid.14022. Epub 2023 Jan 30. PMID: 36714983 6. Sharma V, Aggarwal D, Sharma AK, et al. An overview on Monkeypox, Current Paradigms and Advances in its Vaccination, Treatment and Clinical Management: Trends, Scope, Promise and Challenges. J Pure Appl Microbiol. 2022;16(suppl 1):3000-3012. doi: 10.22207/JPAM.16.SPL1.21 7. Sharma V, Panwar A, Garg VK, et al. Tecovirimat as a Potential Bioavailable Iinhibitor against MPXVgp158 Established through Molecular Dynamic Simulations and Docking Studies. J Pure Appl Microbiol. 2022;16(suppl 1):3168-3178. doi: 10.22207/JPAM.16.SPL1.13 8. Chandran D, Nandanagopal VG, Gopan M, et al. Major Advances in Monkeypox Vaccine Research and Development â An Update. J Pure Appl Microbiol. 2022;16(suppl 1):3083-3095. doi: 10.22207/JPAM.16.SPL1.0 9. Chandran D, Hridya P, Prasanth D, et al. Changing Patterns in the Spread of Human Monkeypox: A Dangerous New Development in Disease Epidemiology. J Pure Appl Microbiol. 2022;16(suppl 1):3106-3118. doi: 10.22207/JPAM.16.SPL1.11  10. Thornhill JP, Barkati S, Walmsley S, et al. Monkeypox virus infection in humans across 16 countries: AprilâJune 2022. N Engl J Med. 2022;387(8):679-691. doi:10.1056/NEJMoa2207323 11. Sigler R, Haidar G. Mpox in immunocompromised patients: with more data, more questions. Transpl Infect Dis. 2023 Feb;25(1):e14023. doi: 10.1111/tid.14023. Epub 2023 Jan 30. PMID: 36714963. 12. Mitjà O, Ogoina D, Titanji BK, Galvan C, Muyembe JJ, Marks M, Orkin CM. Monkeypox. Lancet. 2023 Jan 7;401(10370):60-74. doi: 10.1016/S0140-6736(22)02075-X. Epub 2022 Nov 17. Erratum in: Lancet. 2022 Dec 3;400(10367):1926. PMID: 36403582; PMCID: PMC9671644."
}
]
},
{
"id": "162329",
"date": "13 Feb 2023",
"name": "Osaretin Christabel Okonji",
"expertise": [
"Reviewer Expertise Infectious diseases"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe abstract section looks quite vague. Some more explicit details are necessary\nPlease the first 2 sentences in the abstract should be revised and get straight to the point about mpox global outbreak that started in 2022 putting immunocompromised individuals at increased risk. Please be consistent- use mpox the new recommended name by the World Health Organization (WHO) in the whole of the manuscript. Please remove the symptoms of mpox in the abstract, move to the introduction section or delete. âThe documented severe signs of monkeypox include widespread rashes with secondary fungal or bacterial skin infections or tissue death (necrosis), intestine obstruction, and difficulties with the heart, lungs, urinary system, and nervous systemâ. It will be good to revise this sentence to: We discussed /reviewed the mpox clinical presentation, available treatment options and current preventive guidelines in immunocompromised patients. âThe current article goes into great detail about monkeypox disease occurring in immunocompromised patients and preventive guidelinesâ.\n\nIntroduction\nFirst sentence: please use the new name recommended by WHO, which is mpox Please include that WHO has recommended the name mpox to reduce negative stigma linked with the previous name. Please include the incubation period of mpox 5â21 days in the first paragraph. It will be good to describe the recent outbreak of mpox in 2022 in that first paragraph, but I noticed overlapping of the symptoms of mpox in the abstract and in the other section of the manuscript. Please start a new paragraph about the epidemiology of mpox- âThe epidemiological history of the mpox revealed that⊠In the epidemiology of mpox please include clade 1 and clade 2 strain. The second paragraph in the introduction is not necessary, please delete or move below- This first paragraph should also highlight the importance of the review- since there are very few reviews conducted on mpox in immunosuppressed patients.\n\nThe ongoing 2022 mpox outbreak\nFirst paragraph, the authors should update the viral strain that are now referred to clade 1 and 2 to be consistent with the World Health Organization recommendation. Also include the case fatality rate of 10.6% for clade 1 and 3.6% for clade 2. Second paragraph: please include clinical characteristics of the recent mpox outbreak such as HIV infection, sexual transmitted disease\nTransmission and replication\nLast paragraph should be moved to the ongoing 2022 outbreak of mpox section and the repetition----- mpox among gays, men to men sex should be taken out. Immunosuppression section is not necessary, I suggest it can be briefly written or taken out.\nMonkeypox in immunosuppressed patients\nPlease update monkeypox to mpox Also state detailed information of clinical presentation of mpox among immunosuppressed individuals with regards to the recent outbreak in 2022. This information you can get from CDC website. Second paragraph- Please move to prevention and treatment section Last paragraph- Please add more studies that have reported the clinical presentations of mpox among immunosuppressed patients, include CDC published findings and other studies from Europe, there are many studies, this is to improve this section because it is an important area of this paper.\n\nManagement and prevention\nThe title needs to be revised: Please change to prevention strategies and countermeasures for immunocompromised individuals. Please include- creating awareness of the risk factors and educating people about the measures they need to take to reduce exposure to mpox- which is the main prevention strategy for mpox. Please move the fourth paragraph to the first paragraph, it should be placed at the first line after prevention strategy. Third paragraph- âIn conclusion- please revise The fifth paragraph should be moved to the first and second paragraph.\nConclusion and future prospect\nPlease indicate further research needed to determine the efficacy of antivirals and also the duration of mpox treatment. Please include mpox vaccine efficacy -is necessary to understand the current vaccine efficacy among immunocompromised individuals from the recent vaccination Please indicate clinical trials that are currently ongoing for mpox particularly if there is any one for immunocompromised individualâs The importance of equitable global distribution of mpox countermeasures and high burden regions/countries should be prioritized. Please the conclusion should be succinctly written, and focus should be on the topic.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": [
{
"c_id": "9498",
"date": "03 Apr 2023",
"name": "Sirwan Ahmed",
"role": "Author Response",
"response": "Authors are very thankful to the reviewer for devoting time for improving the quality of the manuscript. The inputs and suggestions from the reviewer have been taken under consideration and have been implemented in the manuscript. The abstract section looks quite vague. Some more explicit details are necessary Answer: The authors have revised the abstract and have made it more updated as well as precise, as suggested by the reviewers. Please the first 2 sentences in the abstract should be revised and get straight to the point about mpox global outbreak that started in 2022 putting immunocompromised individuals at increased risk. Answer: The authors have revised the abstract and have made it more updated as well as precise, as suggested by the reviewers. Please be consistent- use mpox the new recommended name by the World Health Organization (WHO) in the whole of the manuscript. Answer: The authors have incorporated the suggestion of reviewer throughout the manuscript. Please remove the symptoms of mpox in the abstract, move to the introduction section or delete. âThe documented severe signs of monkeypox include widespread rashes with secondary fungal or bacterial skin infections or tissue death (necrosis), intestine obstruction, and difficulties with the heart, lungs, urinary system, and nervous systemâ. Answer: The authors have revised the abstract, as suggested by the reviewers. It will be good to revise this sentence to: We discussed /reviewed the mpox clinical presentation, available treatment options and current preventive guidelines in immunocompromised patients. âThe current article goes into great detail about monkeypox disease occurring in immunocompromised patients and preventive guidelinesâ. Answer: The authors have revised the abstract, as suggested by the reviewers Introduction First sentence: please use the new name recommended by WHO, which is mpox Answer: The mistake has been rectified by the authors. Please include that WHO has recommended the name mpox to reduce negative stigma linked with the previous name. Answer: The mistake has been rectified by the authors. Please include the incubation period of mpox 5â21 days in the first paragraph. Answers: The authors have revised the âIntroductionâ section It will be good to describe the recent outbreak of mpox in 2022 in that first paragraph, but I noticed overlapping of the symptoms of mpox in the abstract and in the other section of the manuscript. Answer: The authors have rectified all these mistakes and updated the manuscript with best of the efforts. Please start a new paragraph about the epidemiology of mpox- âThe epidemiological history of the mpox revealed that⊠Answer: The authors have incorporated the suggestion of the reviewer. In the epidemiology of mpox please include clade 1 and clade 2 strain. Answer: The authors have updated the manuscript as per the suggestion of the reviewer. The second paragraph in the introduction is not necessary, please delete or move below- Answer: The authors have updated the manuscript as per the suggestion of the reviewer. This first paragraph should also highlight the importance of the review- since there are very few reviews conducted on mpox in immunosuppressed patients. Answer: The authors have updated the manuscript as per the suggestion of the reviewer. Mention about the ongoing 2022 mpox outbreak Answer: The authors have updated the manuscript as per the suggestion of the reviewer. First paragraph, the authors should update the viral strain that are now referred to clade 1 and 2 to be consistent with the World Health Organization recommendation. Also include the case fatality rate of 10.6% for clade 1 and 3.6% for clade 2. Answer: The authors have updated the manuscript as per the suggestion of the reviewer. Second paragraph: please include clinical characteristics of the recent mpox outbreak such as HIV infection, sexual transmitted disease Answer: The authors have updated the manuscript as per the suggestion of the reviewer. Transmission and replication Last paragraph should be moved to the ongoing 2022 outbreak of mpox section and the repetition----- mpox among gays, men to men sex should be taken out. Answer: The authors have revised the manuscript and rectified the mistakes as per the suggestion of the reviewer. Immunosuppression section is not necessary, I suggest it can be briefly written or taken out. Monkeypox in immunosuppressed patients Answer: Dear reviewer, we believe it is preferable to include this part; kindly accept our suggestion. Please update monkeypox to mpox Answer: The authors have rectified the mistakes as per the suggestion of the reviewer. Also state detailed information of clinical presentation of mpox among immunosuppressed individuals with regards to the recent outbreak in 2022. This information you can get from CDC website. Answer: The authors have revised the manuscript as per the suggestion of the reviewer. Second paragraph- Please move to prevention and treatment section Answer: The authors have revised the manuscript as per the suggestion of the reviewer. Last paragraph- Please add more studies that have reported the clinical presentations of mpox among immunosuppressed patients, include CDC published findings and other studies from Europe, there are many studies, this is to improve this section because it is an important area of this paper. Answer: The authors have revised the manuscript as per the suggestion of the reviewer. Management and prevention The title needs to be revised: Please change to prevention strategies and countermeasures for immunocompromised individuals. Answer: The authors have revised the manuscript as per the suggestion of the reviewer. Please include- creating awareness of the risk factors and educating people about the measures they need to take to reduce exposure to mpox- which is the main prevention strategy for mpox. Answer: The authors have revised the manuscript as per the suggestion of the reviewer Please move the fourth paragraph to the first paragraph, it should be placed at the first line after prevention strategy. Answer: The authors have revised the manuscript as per the suggestion of the reviewer Third paragraph- âIn conclusion- please revise Answer: The authors have revised the manuscript as per the suggestion of the reviewer The fifth paragraph should be moved to the first and second paragraph. Answer: The authors have revised the manuscript as per the suggestion of the reviewer Conclusion and future prospect Please indicate further research needed to determine the efficacy of antivirals and also the duration of mpox treatment. Answer: The authors have revised the manuscript as per the suggestion of the reviewer Please include mpox vaccine efficacy -is necessary to understand the current vaccine efficacy among immunocompromised individuals from the recent vaccination Please indicate clinical trials that are currently ongoing for mpox particularly if there is any one for immunocompromised individualâs Answer: The authors have revised the manuscript as per the suggestion of the reviewer The importance of equitable global distribution of mpox countermeasures and high burden regions/countries should be prioritized. Answer: The authors have revised the manuscript as per the suggestion of the reviewer Please the conclusion should be succinctly written, and focus should be on the topic. Answer: The authors have revised the manuscript as per the suggestion of the reviewer"
}
]
},
{
"id": "162331",
"date": "21 Feb 2023",
"name": "Dattatreya Mukherjee",
"expertise": [
"Reviewer Expertise Public Health",
"Infectious Disease"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank You for Giving me the opportunity for reviewing the manuscript. This is a very nicely described article. My comments are given below:\n1. Introduction is written in detail. In the introduction please add about the MPOX in immunocompromised patient (in short). If there is any published cases on it, then authors can write it in short in the introduction part.\n\n2. \"The first case of Mpox was reported by the United Kingdom Health Security Agency (UKHSA) in the United Kingdom.\" Please add a reference.\n\n3. An image of mechanism can be added at Transmission and replication part. (Pathophysiology).\n4. In Ongoing MPXV outbreak 2022 part, one image of world map can be added with the color differentiation of cases.\n\n5. In HIV, can we see CD4 count before injecting the vaccine? In that case, in what CD4 count, we can give vaccine? Is there any study on it? If not this paper can recommend this idea for the future. (If authors support my concept of CD4).\n6. Conclusion and future aspects are written very well.\n\n7. The paper has a good discussion on Mpox Vaccine. If authors agree my idea about CD4, can add as a recommendations\n8. Is it possible to have an info graph on the topic? The Clinicians will get to know the treatment procedure quickly in ward. Like if an immunocompromised patient got admitted with Mpox. How the treatment should be given. What are the drug of choices. PCR is the test of Choice, please add that also in the management and info graph parts.\n\nThis is a nicely written and detail review on this topic. Information are written nicely with proper citations. This is a much needed discussion and the paper has filled that void successfully. This paper will be very much helpful for the scientific societies.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "9499",
"date": "03 Apr 2023",
"name": "Sirwan Ahmed",
"role": "Author Response",
"response": "This is a nicely written and detail review on this topic. Information are written nicely with proper citations. This is a much-needed discussion and the paper has filled that void successfully. This paper will be very much helpful for the scientific societies. Answer: Authors are very thankful to the reviewer for devoting time for improving the quality of the manuscript. The inputs and suggestions from the reviewer have been taken under consideration and have been implemented in the manuscript. 1. Introduction is written in detail. In the introduction, please add about the MPOX in immunocompromised patient (in short). If there is any published cases on it, then authors can write it in short in the introduction part. Answer: The authors have carefully revised âIntroductionâ section as per the suggestions of reviewer. 2. \"The first case of Mpox was reported by the United Kingdom Health Security Agency (UKHSA) in the United Kingdom.\" Please add a reference. Answer: The appropriate reference (as given below) is added, as suggested by reviewer. Ward T, Christie R, Paton R S, Cumming F, Overton C E. Transmission dynamics of monkeypox in the United Kingdom: contact tracing study BMJ 2022; 379 :e073153 doi:10.1136/bmj-2022-073153 3. An image of mechanism can be added at Transmission and replication part. (Pathophysiology). Answer: The authors have revised the manuscript. However, the pathophysiology is still unknown accurately. 4. In Ongoing MPXV outbreak 2022-part, one image of world map can be added with the color differentiation of cases. Answer: The authors have carefully revised as per the suggestions of reviewer. 5. In HIV, can we see CD4 count before injecting the vaccine? In that case, in what CD4 count, we can give vaccine? Is there any study on it? If not this paper can recommend this idea for the future. (If authors support my concept of CD4). Answer: The authors have carefully revised as per the suggestions of reviewer. 6. The paper has a good discussion on Mpox vaccine. If authors agree my idea about CD4, can add as a recommendations Answers: Whatever scientific literature is available in the databases have been added. 7. Is it possible to have an info graph on the topic? The Clinicians will get to know the treatment procedure quickly in ward. Like if an immunocompromised patient got admitted with Mpox. How the treatment should be given. What are the drug of choices. PCR is the test of Choice, please add that also in the management and info graph parts. Answer: Dear reviewer, Thanks again for your kind suggestion. We described this information in the figure 2."
}
]
}
] | 1
|
https://f1000research.com/articles/12-127
|
https://f1000research.com/articles/12-235/v1
|
02 Mar 23
|
{
"type": "Review",
"title": "Neurological involvement, immune response, and biomarkers in Kawasaki disease along with its pathogenesis, therapeutic and diagnostic updates",
"authors": [
"Omniat Amir",
"Priyadarshi Prajjwal",
"Pugazhendi Inban",
"Srikanth Gadam",
"Soumya Aleti",
"Rayyan Rafik Sunasra",
"Karan Gupta",
"Mustafa Elhag",
"Mohammed Mahmoud",
"Omklthoum Alsir",
"Priyadarshi Prajjwal",
"Pugazhendi Inban",
"Srikanth Gadam",
"Soumya Aleti",
"Rayyan Rafik Sunasra",
"Karan Gupta",
"Mustafa Elhag",
"Mohammed Mahmoud",
"Omklthoum Alsir"
],
"abstract": "Kawasaki disease is an acute, febrile disease that is not typically fatal if treated and affects infants and children more commonly. More than 80% of the afflicted patients are under the age of four. This disease most commonly affects coronary arteries. In a minority of cases, Aneurysms can burst or produce thrombosis, and they can cause infarction. The distinctive redness in the palms and soles of the feet might result from a delayed-type hypersensitivity reaction to a cross-reactive or recently discovered antigen (s). Autoantibodies against epithelial cells and smooth muscle cells are produced as a result of subsequent macromolecule synthesis and polyclonal white blood cell activation, which intensifies the redness. Kawasaki disease's clinical manifestations range from oral skin disease to the blistering of the mucosa, symptoms involving the hands and the feet, skin disease of the palms and soles, a desquamative rash, and cervical lymphatic tissue enlargement (so it is also referred to as tissue layer lymphatic tissue syndrome). Most untreated patients develop some vessel sequelae, from well-organized coronary inflammation to severe arterial blood vessel dilatation to giant artery aneurysms with rupture or occlusion, infarction, and thrombosis. With human gamma globulin administration, reasonable standards of medical care, and the use of analgesics, the speed of symptomatic progression and inflammatory artery changes are reduced. In this review, we have covered the immunology of Kawasaki disease, its biomarkers, and the neurological manifestations of this multisystem illness. We have also included a discussion on its pathogenesis, diagnosis, and treatment.",
"keywords": [
"Kawasaki",
"Strawberry Tongue",
"Rash",
"Inflammation"
],
"content": "Introduction\n\nDr. Tomisaku Kawasaki originally recognized Kawasaki disease (KD) as a spontaneous lymphoid tissue illness in 1967. It is an abrupt, widespread inflammation with unclear causes that primarily affects newborns and young children. Japan has a high prevalence of KD, with 264.8/1,000,000 children under 5 receiving treatment there in 2012. A major essential KD consequence that affects exactly 25% of untreated individuals is arterial problems.1 Combining KD designation patterns shows seasonality, with incidence peaks that vary based on demographic studies.\n\nIt should be noted that this sickness typically peaks in the winter and spring when illnesses caused by metabolic infectious agents coincide with these seasons. Compared to Japan, where the incidence rate of KD is 330 per 100,000 children aged 0â4 years annually, the United States reports a hospitalization rate of 20 per 100,000 children aged 0â4 years. White Americans and children of Asian origin living in Japan are most at risk, whereas Pacific and Asian heritage residing in the United States are impacted at a moderate incidence. Therefore, any genetic or ethnic origin might potentially impact the etiology of KD.2 Kawasaki disease, being a multisystem illness, can have manifestations in various systems of the body including the central nervous system. The central nervous system manifestations include seizures, facial paralysis, hemiplegia, myositis, etc. It is important to ensure the correct diagnosis and commence proper treatment in order to prevent complications like cerebral infarction or aneurysm. The purpose of this review is to give an overview of the pathogenesis, immunology, and diagnostic biomarkers of KD and help healthcare individuals recognize it early to prevent any complications.\n\n\nMethods\n\nBetween January 1991 and December 2021, sources for this in-depth study were found using searches of PubMed, Google Scholar, Scopus, and the Cochrane Library, as well as references from pertinent papers. âKawasaki,â âStrawberry Tongue,â âRash,â âInflammation,â and âJapanâ were utilized as search phrases along with the conjunctions âORâ and âAND.â Articles in the English language, from the previous 30 years, and those in the pediatric and adult population were included after prescreening with the âHuman studies onlyâ criteria. Titles and abstracts were examined one by one to only include research that was pertinent to the subject. PRISMA guidelines were implemented for the same (Figure 1). Inclusion and exclusion criteria are mentioned in Table 1.\n\n\nDiscussion\n\nSince 1970, countrywide surveys in Japan have been conducted to determine the frequency and incidence of KD. Seventy percent of all KD cases occur in children under three, according to the age distribution at the beginning of the disease, which peaks between 9 and 11 months old. According to the most recent study, more than 2,40,000 registered patients are there overall of this illness. In Japan, the number of youngsters with KD has been rising.3 The surveys also revealed the following: (i) there have been three previous nationwide epidemics; (ii) there hasn't been a recent nationwide epidemic, but there is a small, localized epidemic that is spreading to the nearby region; (iii) the number of patients rises in the winter and falls in the summer; and (iv) Compared to non-siblings, siblings have a far higher chance of transmitting an infection. There are 220 instances per 100,000 children under the age of five in Japan, compared to 100 per 100000 on the peninsula. The prevalence is 10-20 times greater in Western countries.3\n\nRacial characteristics are more important than geographic ones in determining the status of KD. Hawaii's mean rate is 40, although racial disparities are evident; the rates for Japanese, Hawaiians, Chinese, Filipinos, and Caucasians are 360, 95, 77, 56, and seven respectively.3 Furthermore, second-generation KD patients are significantly more numerous than expected, and the relative risk for siblings is ten times higher than expected. A solution to unlocking the riddle of KD may lie in the discovery of genetic elements linked to individual status or radically distinct occurrences among races.3\n\nThe prevalence of the second case illness of Kawasaki disease/mucocutaneous lymph node syndrome among 1788 siblings of children with the illness was calculated using information from questionnaires sent to members of the Japanese Association of Parents of Children with the Syndrome. When the first case in a family occurred at one-year intervals, the general second-case rate for siblings was 2.1%, compared to a corresponding overall incidence of roughly 0.19% in Japan's general population of children ages 0 to 4 during the epidemic year 1982. It was 8.4% for siblings under one year old and 9.3% for those between 35 and two years old. When the initial instances occurred, more than 54.1% of the second cases had already developed in ten days or fewer.4\n\nIn a study of 169 children in an urban centre county, environmental and alternative factors caused the KD. Higher rainfall and lower temperatures were associated with a higher incidence of KD, therefore it appeared that climatic conditions might also be important. The authors were able to control for several seasonality factors since the same connections were validated in the Japanese population using data from thirteen years' worth of countrywide surveys. KD has been proved to be associated with Tropospheric wind as well, with substantial changes in a pattern resembling peaks in KD incidence. A follow-up study suggested that some plant species or other environmental contaminants may be carried by easterly winds coming from northern China and cause KD. Four individuals were found to have evidence of viral infection likely related to KD after numerous considerations of typical background pathology as probable contamination with this sensitive approach. Bocavirus, rhinovirus, infantile paralysis virus, and contagion virus were the illnesses that were known. The number of factor variations linked to KD has significantly decreased as a result of genome-wide association studies.\n\nIn turn, these discoveries have opened up fresh perspectives on the pathophysiology of KD. Examples include one polymorphism within the FCGR2A factor and two susceptibleness loci that were known from one of the most significant social cohort studies. The FcRIIA receptor, a low-affinity Ig Fc receptor, is encoded by this factor. Immune cells, particularly phagocytes, express FcRIIA in a healthy manner. Following this, many FCGR2A polymorphisms have been linked to KD susceptibility in a variety of ethnic groups. Given the relevance of IV IG in the treatment of KD and the ongoing lack of a thorough understanding of the mechanism behind IV IG efficacy in KD, the connection of FCGR2A with risk for KD is particularly intriguing. Notably, IVIG resistance is correlated with high levels of FcRIIA messenger RNA expression. The second locus discovered in this study was ITPKC, a factor previously related to KD susceptibility in a disproportionately Japanese population. The ITPKC factor encodes B-1,4,5-triphosphate-3-kinase C, an association in nursing protein that reduces the calcium response in immune cells. Mixed inflammatory infiltrates comprising PMNLs, eosinophils, macrophages, and lymphocytes are detected affecting the tissue layer of the main coronary arteries in the early stages of the illness (about the first 10 days). The media is relatively unaffected. This severe inflammatory stage is characterized by perivasculitis, which also affects the blood vessels and microvessels.5\n\nEach innate and resolving immune cell gets activated and infiltrated into the arterial blood vessel wall as part of the disease's progressive response. A tube-shaped structure pathology has been categorized into three successive related with the diseased processes on the basis of examinations of post-mortem tissue from individuals with this disease. Within the first two weeks of the sickness, necrotizing redness appears and is caused by neutrophil infiltrations that gradually obliterate the media, tissue layer, and some deeper tissue layers of the arterial blood vessels.6 Leucocyte infiltration, however, was rapidly followed by white blood cell infiltrates throughout a series of six specimens, and then mixed white blood cell and plasmacytic infiltrates became undeniable later, close to day nineteen of the illness. Along with induration of the arteries 39 plaque development, distinct nodular infiltrates have also been seen; however, they tend to develop at later time periods (>3 weeks). T cells, macrophages, B cells, and IgM+ plasma cells, which were more prevalent than IgA+ plasma cells, made up these infiltrates.7 The number of IL-1-related genes and raised levels of IL-1 pathway proteins in plasma provide strong evidence that the lymphokine (IL-1) pathway is activated in KD. The IL-6/T helper TH-17 axis 21 and the IL-12/interferon-gamma axis are two major protein clusters identified in KD. Naive T cells are polarised toward a Th-17 composition by IL-6 and remodelling protein beta (TGF), 21 which results in these cells penetrating the vessel wall and altering the protein profile.8 A CD4:CD8 ratio of 2.3-2.7 in the peripheral blood in acute KD indicates a substantial reversal of the CD4:CD8 ratio in the disease's principal target tissue.9\n\nPatients will have a fever of at least 100.4°F for at least five days. The duration of a fever without treatment might reach eleven days. There are at least four of the following symptoms in addition to the fever:\n\ni) A rash covering the body's extremities and the trunk.\n\nii) The palms and soles of the feet become red and edematous. Within the second and third weeks, the skin on the fingers and toes begins to peel.\n\niii) Red eyes that might be light-sensitive.\n\niv) Mouth, lip, and throat irritation and inflammation. A âstrawberryâ tongue is recognizable because it has larger style buds and is joltingly red.\n\nAbdominal discomfort is a potential symptom for patients. Only a tiny percentage of individuals experience transient inflammatory diseases that cause knee and hip pain and swelling. If a newborn has fever and inflammation but none of the other signs are present, incomplete illness should be taken into consideration.10 Additionally, there may be bilateral non-exudative conjunctivitis.11\n\n\n\ni) Myocarditis, endocarditis, pericarditis.\n\nii) Congestive heart failure\n\niii) The CNS, RS, musculoskeletal system, and other bodily systems may all be affected by Kawasaki illness.\n\niv) Coronary artery aneurysms, calcifications, strictures, and dysfunction brought on by scarring of the leaflets or progressively dilated artery roots.12\n\nv) Coronary artery thrombosis-induced occlusion.13\n\nA medical expert should evaluate the child and look for symptoms and indications that are typical with KD. Although these tests aren't specific for KD, blood testing and other serological tests can be performed to rule out alternative disorders and the presence of inflammation. The coronary arteries and the way the valves are working may both be clearly seen in an Ultrasonography scan. This could help in identifying the disease early to avoid any sequelae.14\n\nThe use of high-dose IV IG is recommended by the most recent management recommendations for mucocutaneous lymph node syndrome. If administered for the first ten days of the illness, IV IG lowers the risk of coronary rubor and aneurysm development. Although the exact methods by which the IV IG therapy lowers inflammatory responses are yet understood, it is believed to have a broad range of activity that targets several immune response pathways. It has been demonstrated that IV IG inhibits macrophages' ability to produce IL-1 in vitro and promotes the assembly of IL-1Ra in mucocutaneous lymph node syndrome. By saturating Fc receptors,15 it also lessens the synthesis of chemokines and pro-inflammatory cytokines as well as the activation of neutrophils, monocytes, macrophages, and T cells.16\n\nIn research, clarithromycin and endogenous antibody were used, and the results revealed that clarithromycin lowers the KD relapse rate. Through its anti-biofilm function, clarithromycin appears to be useful in preventing KD recurrence by preventing the release of bioactive chemicals. Clarithromycin may thus be beneficial in treating people with the acute form of KD as well as their siblings to stop the disease from spreading to them.17 There were fewer refractory KD patients in the combination therapy cluster than in the IV IG alone cluster in a trial of two groups, one receiving an anti-TNF drug along with IV IG, and the other receiving IV IG alone. Patients with KD who received combination therapy had better results, shorter hospital stays and fever durations, and less arterial dilatation.18 Children with KD can get safe and effective antithrombotic treatment using clopidogrel in combination with a low-dose anodyne.19 The combination of the aforementioned medication with a lipid-lowering drug with immunomodulatory effects has the potential to be beneficial for children with acute mucocutaneous lymph node syndrome and early symptoms of coronary artery damage.20\n\nWhen a child is detected with Kawasaki illness, the degree and severity of coronary arteries damage at diagnosis and during evaluation are the main factors determining prognosis. The most common cause of death is coronary artery disease leading to myocardial infarction brought on by the blockage of the vessel, and the actual case-fatality rate in Japan and the US is less than 0.2%. A thorough risk classification methodology is provided by the AHA standards when it comes to the assessment, cure, and management of KD,21 which can be used as follow-up counseling. Five risk groups are used in the classification scheme, which uses both absolute luminal measurements as well as Z scores. When the coronary arteries are not involved, the danger level is 1 (Z score 2). Both during the acute sickness and 6â8 weeks after the disease started, these individuals are examined with echocardiograms. These patients resemble people without a KD diagnosis in terms of risk assessment.22 As long as, in these individuals, the risk classification does not alter negatively, ASA medication can be stopped in this group. Risk category 5 having large or massive aneurysms is the category with the greatest risk. These patients need considerably more frequent cardiac monitoring and may potentially need to take anticoagulants if their aneurysms continue to develop as described above.23\n\nEarly diagnosing is vital for the prompt establishment of intravenous gamma globulin medical treatment in KD.24 The ordinarily used inflammatory markers, (ESR), (CRP), and total leukocyte counts (TLC) lack specificity for KD.25 CASP5 (Caspase 5) and CR1(Complement C3b/C4b Receptor 1) genes were shown to probably discriminate KD from alternative similar diseases and additionally from healthy folks.26 Solely four laboratory markers at presentation correlate with CAA development in KD: lower platelets, high neutrophils, high lymphocytes, and high C-reactive protein.27 So as to understand the diagnostic serum biomarkers for KD, a previous study explored serum KD-related proteins, that were differentially expressed throughout the acute and recovery phases of two patients by mass chemical analysis (MS). The degree of 3 of those proteins, particularly lipopolysaccharide-binding macromolecule (LBP), leucine-rich alpha-2-glycoprotein (LRG1), and angiotensinogen (AGT), were higher in acute section patients. Also, the amount of retinol-binding macromolecule four (RBP4) was ablated.28 Lipoprotein receptor eleven (LR11) may be a member of the beta-lipoprotein receptor family, which is expressed markedly in membrane vascular SMCs and secreted in a very soluble type (sLR11). sLR11 has been recently known as a possible vascular lesion biomarker and is reportedly elevated in patients with arterial blood vessel chronic lesions, however, there's no description of sLR11 in acute KD. sLR11 also can mirror the state of acute KD and could be a biomarker for patient response to IVIG medical aid.29 Differentially expressed proteins (DEPs) in KD as compared to traditional controls, additionally created the functional annotation and protein interaction (PPI) networks in KD and respiratory disease. Forty-three and sixty-two DEPs were known in KD and respiratory disease, respectively. Serinehydroxymethyl transferase may be an important hub macromolecule of the KD-specific PPI network.30 Serum ferritin is understood to be a helpful biomarker for the prediction of IVIG-nonresponsive KD, coronary artery abnormalities (CAAs), and macrophage activation syndrome (MAS) tendencies.31 In a study, carboxyterminal propeptide of procollagen type I (PIPC), soluble suppressor of tumorigenicity II, galectin-3 (Gal-3), and growth-differentiation factor-15 were associated with the late convalescent blood cultures from a group of sixteen KD patients, who had an ejection fraction of less than 55% on their initial echocardiogram test. Results were analyzed and compared with blood samples from similar sex and age KD individuals having initial ejection fraction of more than 60%. According to the univariate analysis of the groups, the median Gal-3 and levels of PIPC within the low ejection fraction cluster were considerably much higher than those within the traditional ejection fraction cluster. Convalescent KD individuals with a history of low ejection fraction throughout the acute sickness had considerably increased levels of Gal-3 and PIPC as compared to that in the KD patients in the second group. These findings indicate the possible development of fibrosis as a late abnormalcy of severe carditis which might develop as sequelae to KD.32 Also, a set of five genes are candidates as biomarkers for KD diagnosing, namely, the HLA-DQB1, HLA-DRA, ZBTB48, TNFRSF13C, and CASD1.33\n\nNeurologic involvement in KD is reported in 1.1% to 3.7% of KD cases.34 It ranges from common symptoms like irritability and lethargy to multi-system involvement such as seizures, myositis, sterile infectious diseases, facial palsy, hemiplegia, and coma. The role of single-photon emission computed imaging (SPECT) is rising in the analysis of cerebral hypoperfusion in KD secondary to cerebral rubor.35 Facial nerve paralysis is predominant in females and most facial paralysis is unilateral, and mostly on the left side.36â37\n\nCerebral infarction may be a rare complication of KD and happens within the acute or subacute stage, A case report in a 5-month-old male patient with KD showed that even after receiving timely intravenous immunoglobulin therapy, he developed coronary artery aneurysms (CAA) and coronary artery thrombosis (CAT) one month later. Medical aid and thrombolytic agents were suggested, however, the childâs parents refused. Fifteen months after KDâs onset, an associated attack of syncope left him with left hemiplegia. CT scans revealed cerebral infarction of the right basal ganglion without hemorrhage.38 Cerebral infarction in KD is usually attributed to (1) inflammatory pathology or occlusion of cervical or intracranial large-sized vessels, or (2) occlusion from hypokinetic cardiac muscle.39\n\nThe neurological manifestations in KD can present as headache, convulsions, somnolence, extreme irritability, signs of membrane irritation, bulging fontanelles, and facial palsy.40 Histopathological studies from postmortem examinations of KD patients have shown neuritis and ganglionitis of bones and peripheral nerves, aseptic meningitis, and meningitis. It's been believed that vasculitis of the vessels supplying the nerves could account for the impact on peripheral nerves. A report of the coincident development of migraine and Raynaudâs development twelve years after the development of KD suggested that KD might result in endothelial tissue pathology dysfunction as a late complication.41\n\nAcute encephalopathy, subdural effusion, and dyssynergia have also been reported.42\n\n\nConclusion\n\nThe diagnosis, immediate care, and long-term therapy of KD are discussed in this article along with updated suggestions. The avoidance of significant coronary artery anomalies is the ultimate objective. The difficulties of an isolated incident of KD throughout childhood are thought to be the cause of the unexpected death or CAD that younger adult patients are continuing to present with. Even though the finest available information and professional opinion were used to construct this review, significant evidence gaps were found. A specific diagnostic test continues to be difficult, and acute management is rather empirical until the etiology and pathophysiology of this condition are identified properly and early. Furthermore, there is no way for us to prohibit KD. A tiny fraction of individuals may develop facial paralysis, cerebral infarction, and hemiplegia. Some may have coronary artery aneurysms when they are first diagnosed or develop them despite the most advanced empiric treatment now available, to which some individuals do not react.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nFukui S, Seki M, Minami T, et al.: Efficacy and safety associated with the infusion speed of intravenous immunoglobulin for the treatment of Kawasaki disease: a randomized controlled trial. Pediatr. Rheumatol. Online J. 2021 Jul 3; 19(1): 107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLo MS: A framework for understanding Kawasaki disease pathogenesis. Clin. Immunol. 2020 May; 214: 108385. Epub 2020 Mar 12. PubMed Abstract | Publisher Full Text\n\nTakahashi K, Oharaseki T, Yokouchi Y: Pathogenesis of Kawasaki disease. Clin. Exp. Immunol. 2011 May; 164(Suppl 1): 20â22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFujita Y, Nakamura Y, Sakata K, et al.: Kawasaki disease in families. Pediatrics. 1992 Oct; 84(4): 666â669. PubMed Abstract | Publisher Full Text\n\nRamphul K, Mejias SG: Kawasaki disease: a comprehensive review. Arch. Med. Sci. Atheroscler. Dis. 2018 Mar 21; 3: e41âe45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoval Rivas M, Arditi M: Kawasaki disease: pathophysiology and insights from mouse models. Nat. Rev. Rheumatol. 2020 Jul; 16(7): 391â405. Epub 2020 May 26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLindquist ME, Hicar MD: B Cells and Antibodies in Kawasaki Disease. Int. J. Mol. Sci. 2019 Apr 13; 20(8): 1834. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNewburger JW, Takahashi M, Burns JC: Kawasaki Disease. J. Am. Coll. Cardiol. 2016 Apr 12; 67(14): 1738â1749. PubMed Abstract | Publisher Full Text\n\nBrown TJ, Crawford SE, Cornwall ML, et al.: CD8 T Lymphocytes and Macrophages Infiltrate Coronary Artery Aneurysms in Acute Kawasaki Disease. J. Infect. Dis. 1 October 2001; 184(7): 940â943. Publisher Full Text\n\nKim KY, Kim DS: Recent Advances in Kawasaki Disease. Yonsei Med. J. 2016 Jan; 57(1): 15â21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim DS: Kawasaki disease. Yonsei. Med. J. 2006 Dec 31; 47(6): 759â772. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGordon JB, Kahn AM, Burns JC: When children with Kawasaki disease grow up: Myocardial and vascular complications in adulthood. J. Am. Coll. Cardiol. 2009 Nov 17; 54(21): 1911â1920. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSivakumar K, Pavithran S: Extensive coronary aneurysms with thrombosis in resistant Kawasaki disease. Pediatr. Cardiol. 2013 Feb; 34(2): 444â446. Epub 2012 Mar 18. PubMed Abstract | Publisher Full Text\n\nde La Harpe M , di Bernardo S , Michaël H, et al.: Thirty Years of Kawasaki Disease: A Single-Center Study at the University Hospital of Lausanne. Front. Pediatr. 2019; 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoval Rivas M, Arditi M: Kawasaki disease: pathophysiology and insights from mouse models. Nat. Rev. Rheumatol. 2020 Jul; 16(7): 391â405. Epub 2020 May 26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPortman MA, Dahdah NS, Slee A, et al.: Etanercept With IVIg for Acute Kawasaki Disease: A Randomized Controlled Trial. Pediatrics. 2019 Jun; 143(6): e20183675. Epub 2019 May 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNanishi E, Nishio H, Takada H, et al.: Clarithromycin Plus Intravenous Immunoglobulin Therapy Can Reduce the Relapse Rate of Kawasaki Disease: A Phase 2, Open-Label, Randomized Control Study. J. Am. Heart Assoc. 2017 Jul 6; 6(7): e005370. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurns JC, Roberts SC, Tremoulet AH, et al.: Infliximab versus second intravenous immunoglobulin for treatment of resistant Kawasaki disease in the USA (KIDCARE): a randomised, multicentre comparative effectiveness trial. Lancet Child Adolesc Health. 2021 Dec; 5(12): 852â861. Epub 2021 Oct 27. Erratum in: Lancet Child Adolesc Health. 2022 Feb;6(2):e5. PubMed Abstract | Publisher Full Text\n\nLiu YL, Wang XM, Chen TT, et al.: Clinical effect and safety of clopidogrel combined with aspirin in antithrombotic therapy for children with Kawasaki disease complicated by small/medium-sized coronary artery aneurysms. Zhongguo Dang Dai Er Ke Za Zhi. 2019 Aug; 21(8): 801â805. Chinese. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTremoulet AH, Jain S, Jone PN, et al.: Phase I/IIa Trial of Atorvastatin in Patients with Acute Kawasaki Disease with Coronary Artery Aneurysm. J. Pediatr. 2019 Dec; 215: 107â117.e12. Epub 2019 Sep 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKato H, Sugimura T, Akagi T, et al.: Long-term consequences of Kawasaki disease. A 10- to 21-year follow-up study of 594 patients. Circulation. 1996 Sep 15; 94(6): 1379â1385. PubMed Abstract | Publisher Full Text\n\nBeiser AS, Takahashi M, Baker AL, et al.: A predictive instrument for coronary artery aneurysms in Kawasaki disease. US Multicenter Kawasaki Disease Study Group. Am. J. Cardiol. 1998 May 1; 81(9): 1116â1120. PubMed Abstract | Publisher Full Text\n\nYale SH, Tekiner H, Yale ES: Tomisaku Kawasaki and Kawasaki disease. Childs Nerv. Syst. 2022; 38: 233â235. Publisher Full Text\n\nRowley AH, Pink AJ, Reindel R, et al.: A study of cardiovascular miRNA biomarkers for Kawasaki disease. Pediatr. Infect. Dis. J. 2014 Dec; 33(12): 1296â1299. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiomarkers for Kawasaki Disease:Clinical Utility and the Challenges Ahead Himanshi Chaudhary, Johnson Nameirakpam, Rajni Kumrah, Vignesh Pandiarajan,Deepti Suri, Amit Rawat and Surjit Singh.\n\nRahmati Y, Mollanoori H, Najafi S, et al.: CASP5 and CR1 as potential biomarkers for Kawasaki disease: an Integrated Bioinformatics-Experimental Study. BMC Pediatr. 2021; 21: 566. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGeorge A, McCormack R: Tulloh RG85(P) Admissions to hospital and awareness of kawasaki disease in england. Arch. Dis. Child. 2018; 103: A35.\n\nKimura Y, Yanagimachi M, Ino Y, et al.: Identification of candidate diagnostic serum biomarkers for Kawasaki disease using proteomic analysis. Sci. Rep. 2017 Mar 6; 7: 43732. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatanabe K, Suzuki H, Jiang M, et al.: Soluble LR11 as a Novel Biomarker in Acute Kawasaki Disease. Circ. J. 2022 May 25; 86(6): 977â983. Epub 2021 Sep 16. PubMed Abstract | Publisher Full Text\n\nHu HM, Du HW, Cui JW, et al.: New biomarkers of Kawasaki disease identified by urine proteomic analysis. FEBS Open Bio. 2018 Dec 20; 9(2): 265â275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQiu Z, Liu HH, Fan GZ, et al.: The clinical implications of serum ferritin in Kawasaki disease: a helpful biomarker for evaluating therapeutic responsiveness, coronary artery involvement and the tendency of macrophage activation syndrome. Arch. Med. Sci. 2021 Dec 8; 18(1): 267â274. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoshino S, Shimizu C, Jain S, et al.: Biomarkers of Inflammation and Fibrosis in Kawasaki Disease Patients Years After Initial Presentation With Low Ejection Fraction. J. Am. Heart Assoc. 2020 Jan 7; 9(1): e014569. Epub 2019 Dec 27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPezoulas VC, Papaloukas C, Veyssiere M, et al.: A computational workflow for the detection of candidate diagnostic biomarkers of Kawasaki disease using time-series gene expression data. Comput. Struct. Biotechnol. J. 2021 May; 19(19): 3058â3068. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJindal AK, Pilania RK, Suri D, et al.: An Adult With Fever and Facial Nerve Palsy: An Unusual Presentation of Kawasaki Disease. J. Clin. Rheumatol. 2020 Aug; 26(5): e142âe143. PubMed Abstract | Publisher Full Text\n\nSato T, Ushiroda Y, Oyama T, et al.: Kawasaki disease-associated MERS: pathological insights frfr SPECT findings. Brain and Development. 2012; 34: 605â608. PubMed Abstract | Publisher Full Text\n\nSun M, Xu X, Li H, et al.: Facial Nerve Paralysis- Rare Neurological Complication of Kawasaki Disease. Biomedical Journal of Scientific & Technical Research, Biomedical Research Network+, LLC. 2020 October; vol. 31(3): pages 24201â24204.\n\nMcDonald D, Buttery J, Pike M: Neurological complications of Kawasaki disease. Arch. Dis. Child. 1998 Aug; 79(2): 198. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang L, Duan H, Zhou K, et al.: Kawasaki Disease Complicated by Late-Onset Fatal Cerebral Infarction: A Case Report and Literature Review. Front. Pediatr. 2021 May 19; 9: 598867. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGitiaux C, Kossorotoff M, Bergounioux J, et al.: Cerebral vasculitis in severe Kawasaki disease: early detection by magnetic resonance imaging and good outcome after intensive treatment. Dev. Med. Child Neurol. 2012 Dec; 54(12): 1160â1163. PubMed Abstract | Publisher Full Text\n\nLiu X, Zhou K, Hua Y, et al.: Neurological involvement in Kawasaki disease: a retrospective study. Pediatr. Rheumatol. Online J. 2020 Jul 14; 18(1): 61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTulloh RMR, Mayon-White R, Harnden A, et al.: Kawasaki disease: a prospective population survey in the UK and Ireland from 2013 to 2015. Arch. Dis. Child. 2019 Jul; 104(7): 640â646. Epub 2018 Aug 13. Erratum in: Arch Dis Child. 2020 Mar;105(3):e5. PubMed Abstract | Publisher Full Text\n\nAbe Y, Ayusawa M, Kawamura K, et al.: A Combination Therapy for Kawasaki Disease with Severe Complications: a Case Report. Open Med (Wars). 2019 Dec 26; 15: 8â13. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "165333",
"date": "07 Mar 2023",
"name": "Alhad Mulkalwar",
"expertise": [
"Reviewer Expertise Internal Medicine",
"Research"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review discusses Kawasaki disease in good detail. The authors have included various subsections such as pathogenesis, neurological signs, immune response, diagnosis, biomarkers, complications, treatment, and prognosis. The article is well-summarised and scientifically accurate. For instance, it mentions the diagnostic criteria such as that patients should have a fever for at least five days of more than 100.4 F. It is usually accompanied by a rash, edematous palms, and soles, reddening of eyes, and strawberry tongue. The cerebral symptoms include infarction, stroke, paralysis, and facial palsy. Some places such as diagnosis and diagnostic criteria could have been described a bit more and be clearer. But, overall the article is well written and the subtopics are properly summarised as mentioned.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
},
{
"id": "165334",
"date": "20 Mar 2023",
"name": "Arpit Mago",
"expertise": [
"Reviewer Expertise immunogenetics and neurological manifestations"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would first like to congratulate the author team for their efforts in bringing a good research paper but I feel certain issues need to be addressed:\nIn the methods, type of research design should be clearly described.\n\nThere is a mismatch in the databases in the PRISMA Flow diagram and the ones mentioned in the text, it needs to be correct. Also the total reports sought for retrieval mentioned are 500 but in the second step only 120 are mentioned to be removed out of 720, so the final number should 600. There should be a clear mention of where the other 100 articles were excluded.\n\nThere is no mention if case reports, letters and editorials which often talk about new advances in case of different diseases were included or not\n\nIn table 1 it's mentioned that animal experiments are also included but in the text it's mentioned that only human studies were included. Please explain this statement. Besides last one, inclusion and exclusion is quite irrelevant and should be removed.\n\nThe discussion is heavily focussed on Japan, it should be more broad and compare the incidence of Kawasaki disease in different continents or at least in most countries that disease is prevalent.\n\nDiscussion is very unstructured. Biomarkers should be talked about under the diagnosis section as it's a component of it and should be clubbed with the treatment under management section. Neurological involvement is also just a separate heading breaking the flow of ideas, it should be properly included under the symptomatology section. Besides the pathology section should be more elaborate with mention of the inconsistencies reported in the pathology in other published articles.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": [
{
"c_id": "9482",
"date": "03 Apr 2023",
"name": "Omniat Amir",
"role": "Author Response",
"response": "Hi, Thank you for your detailed and positive review. The methods section and Prisma diagram have been updated to include the details more clearly. Although previously too, the incidence of KD in some countries were talked about, now one more paragraph and a figure have been added comprehensively discussing the incidence of KD in various other countries. The biomarker subsection is shifted below the diagnosis subsection, and the Neurological involvement subsection is shifted below the symptoms subsection to facilitate easy understanding in the new version."
}
]
}
] | 1
|
https://f1000research.com/articles/12-235
|
https://f1000research.com/articles/11-962/v1
|
19 Aug 22
|
{
"type": "Research Article",
"title": "Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in nontuberculous mycobacteria isolates from Kenya",
"authors": [
"Zakayo Mwangi",
"Grace Naeku",
"Marianne Mureithi",
"Frank Onyambu",
"Wallace Bulimo",
"Grace Naeku",
"Marianne Mureithi",
"Frank Onyambu",
"Wallace Bulimo"
],
"abstract": "Background: Nontuberculous mycobacteria (NTM) treatment constitutes a macrolide-based antibiotic regimen in combination with aminoglycosides for Rapid-Growing mycobacteria (RGM), and rifampicin for Slow-Growing mycobacteria (SGM). Mutations in the anti-NTM drug target regions promote NTM evolution to mutant strains that are insusceptible to NTM drugs leading to treatment failure. We, therefore, described the mutation patterns of anti-NTM drug target genes including rrl, rrs, and rpoB in NTM isolates from Kenya. Methods: We carried out a cross-sectional study that included 122 NTM obtained from the sputum of symptomatic tuberculosis-negative patients in Kenya. All the 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced for rrs, and the 68 SGM were sequenced for rpoB genes using ABI 3730XL analyzer. The obtained sequences were aligned to their wild-type reference sequences for each gene using Geneious then mutations were identified. Pearson chi-square at 95% confidence interval tested the association of NTM to mutation patterns for each gene. Results: Twenty-eight (23%) of the NTM were resistant to at least one of the antibiotics used in the macrolide-based treatment. Twelve (10.4%) of NTM were macrolide resistant, with 7(58.3%) of RGM and 5(41.7%) of SGM having mutations in the rrl gene. For ten (83.3%) NTM, mutations were found at position 2058, while for two (16.6%) NTM, mutations were found at position 2059. Six (11.1%) of the 54 RGM exhibited mutations in the aminoglycoside target gene rrs at location 1408. Ten (14.7%) of the 68 SGM were resistant to rifampicin, with 40 percent having mutations at codon 531 in the rpoB gene.\n\nConclusion: We demonstrated a significant level of drug resistance for macrolides, aminoglycosides and rifampicin in NTM isolated from symptomatic TB negative patients in Kenya.",
"keywords": [
"Nontuberculous mycobacteria",
"slow-growing mycobacteria",
"rapid-growing mycobacteria",
"macrolides",
"aminoglycosides",
"rifampicin"
],
"content": "Introduction\n\nNon-tuberculous Mycobacteria (NTM) are a group of about 170 Mycobacteria species that do not include Mycobacterium tuberculosis and Mycobacterium leprae (Falkinham, 2017; Koh et al., 2006; Peixoto et al., 2020; Sam et al., 2020; Simons et al., 2011). In mycobacterial culture, NTM varies in their growth characteristics, with rapid growing NTM (RGM) forming visible colonies within seven days of incubation and slow-growing NTM (SGM) taking up to fourty days. SGM include Mycobacterium avium complex (MAC), Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium malmoense, and Mycobacterium xenopi, whereas RGM comprise species from the Mycobacterium abscessus and Mycobacterium fortuitum complexes (Alffenaar et al., 2021). In human infections, identifying NTM is crucial for determining clinically relevant species and determining the best treatment plan (Chalmers et al., 2019; Mwangi et al., 2022).\n\nNTM treatment consists of a macrolide-based antibiotic regimen, such as clarithromycin or azithromycin, combined with other selected antibiotics that work synergistically to disrupt NTM metabolic processes and growth (Falkinham, 2018). In addition to macrolides, the antibiotic of choice is largely determined by the infecting NTM, its growth rate, and the intricacy of the mycolic acid cell wall (Goswami et al., 2016). Rifampicin and ethambutol are also used in SGM treatment, while aminoglycosides, cefoxitin, imipenem, or tigecycline are used in RGM treatment (Brown-Elliott et al., 2012; Saxena et al., 2021). Due to the slow growth of NTM compared to other bacterial infections, antimicrobial combination therapy is strongly recommended in NTM treatment to avoid the development of drug resistance (Falkinham, 2018; Pharmd et al., 2019).\n\nAnti-mycobacterial drugs attach to their binding sites with a high affinity, preventing the target gene product from functioning normally (Alffenaar et al., 2021). Changes in the structure of the target regions caused by mutations, on the other hand, interfere with the medications' ability to attach to them, resulting in antibiotic resistance. As a result, determining the antibiotic resistance profile of NTM is critical for determining an effective treatment strategy for a specific NTM infection (Goswami et al., 2016; Nasiri et al., 2017).\n\nAcquired resistance to anti-NTM drugs develops due to mutations in the NTM drug target regions, subsequently promoting NTM evolution to mutant strains that are insusceptible to anti-NTM drugs (Huh et al., 2019; Munita & Arias, 2016; Nasiri et al., 2017; Pharmd et al., 2019; Saxena et al., 2021). Prolonged exposure to NTM antibiotics, as seen in the lengthy NTM regimen, sub-optimal administration of anti-NTM drugs, as seen in patients who do not adhere to the regimen or who are lost to follow up, and incorrect prescription for NTM infection due to NTM misdiagnosis all promote mutation in the drug target regions (Gopalaswamy et al., 2020; Munita & Arias, 2016; Zhou et al., 2020).\n\nMacrolides inhibit protein synthesis by binding to the peptide exit tunnel of ribosomes, hence preventing the growing peptide chain from exiting the peptidyl transferase center of the ribosome (Hansen et al., 2002). Mutation of the rrl gene at positions A2058 and A2059 accounts for 80-100% of the macrolide resistance in NTM. Macrolide resistance can also be conferred by the erm (41) gene which encodes a ribosomal methyltransferase that methylates the rrl thus blocking the drug-binding site of the macrolide (Bastian et al., 2011; Huh et al., 2019).\n\nAminoglycosides inhibit protein synthesis by binding the bacterial 30S ribosomal subunit interfering with bacterial protein translation and leading to cell death (Saxena et al., 2021). Drug resistance to aminoglycosides is associated with modification in the rrs gene mostly observed as point mutation at position 1408 (Brown-Elliott et al., 2013). In addition, mutations at positions 1406, 1409, and 1491 have been shown to confer resistance to aminoglycosides in some NTM (Olivier et al., 2017).\n\nRifampicin is a key drug in treating mycobacterial diseases including those caused by M. tuberculosis (WHO, 2014). It inhibits the synthesis of Ribonucleic acid (RNA) by binding to the Ã-subunit of the RNA polymerase that is encoded by rpoB gene. Most rifampicin-resistant mycobacteria have mutations occurring in an 81-bp rifampicin resistance determining region (RRDR) within the rpoB gene. Mutations in this region account for 95% of rifampicin resistance in mycobacteria. The commonly observed mutations within the RRDR of rpoB are often seen at codons 526, and 531 (Li et al., 2016). M. kansasii has also shown mutation conferring resistance to rifampicin at codons 513 and 516, while MAC also shows resistance to rifampicin with mutations outside the RRDR at codon 626 (K626T) (Ramasoota et al., 2006).\n\nDetection of drug resistance in NTM can be carried out by drug susceptibility testing (DST) through broth microdilution, (Litvinov et al., 2018; Park et al., 2020), sequencing for mutations in the rrl, rrs and rpoB genes (Brown-Elliott et al., 2012; Huh et al., 2019; Saxena et al., 2021), or by using GenoType NTM-DR test (Hain, Lifescience, Nehren, Germany) which is a line probe assay (LPA) that detects resistance to macrolides and aminoglycosides (Bouzinbi et al., 2020).\n\nWe, therefore, investigated drug resistance in NTM by describing the mutation patterns in rrl, rrs, and rpoB genes for macrolides, aminoglycosides and rifampicin respectively, in NTM isolated from symptomatic TB negative patients from Kenya.\n\n\nMethods\n\nWe carried out a cross-sectional study that included 122 NTM identified by mycobacterial culture and hsp65 targeted sequencing from the sputum of symptomatic TB-negative patients (Mwangi et al., 2022). The samples were obtained from the National Tuberculosis Reference Laboratory (NTRL) in Kenya between January to November 2020. All the 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced for rrs, and the 68 SGM were sequenced for rpoB genes using ABI 3730XL analyzer (Applied Biosystems, Foster City, California, USA).\n\n\nLaboratory procedures\n\nThe sputum samples were decontaminated using the N-acetyl-L-cysteine 2% NaOH (NALC-NaOH) procedure, then inoculated into Mycobacteria Growth Indicator Tube (MGIT) and Lowestein-Jenseen (LJ) medias, incubated at 37°C and monitored for growth for up to eight weeks respectively. At the same time, sputum smears were prepared, air dried, heat fixed then fluorochrome stained with auramine O where mycobacteria appeared as bright yellow fluorescent rods when viewed under a light emitting diodes (LED) microscope.\n\nThe culture growth in MGIT and LJ underwent the Mtb identification testing using the SD Bioline TB Ag MPT64 assay (capilia) (Standard Diagnostics, Yongin-si, Gyeonggi-do, Republic of Korea) and capilia positive samples were excluded from the study. The capilia negative samples underwent ZN microscopy with presence of AFB indicating a possible NTM.\n\nMycobacterial DNA was extracted from 500 ÎŒL of re-suspended colonies using using GenoLyse® (Hain Lifescience, Nehren, Germany) according to the manufacturer's instructions. Briefly, 100 ul of lysis buffer (A-LYS) buffer was added to each cryovial containing the resuspended colonies and incubated for five minutes at 95oC after which 100 ul neutralization buffer (A-NB) was added and centrifugation done at 5000G for ten minutes. The supernatant was transferred to a newly labelled cryovial awaiting PCR.\n\nConventional PCR targeting rrl, rpoB and rrs genes of NTM were conducted using the Horse-Power⢠Taq DNA Polymerase MasterMix (Canvax, Córdoba, Spain) in a final reaction volume for each gene of 13 ÎŒl comprising 6.25 ÎŒl of 2X Horse-Power⢠Taq DNA Polymerase MasterMix, 2.5 ÎŒl DNA template, 0.25 ÎŒl of each of both forward and reverse primers (Table 1) at a final concentration of 10 pmoles, and 3.75 ÎŒl of nuclease-free water to make up the reaction volume. The PCR assays were carried out with a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA) (Table 1). Thermal cycling conditions for rrl were as follows: one cycle of 95°C for five minutes, 35 cycles of 95°C for one minute, 55°C for one minute, 72°C for one minute, and a final extension for ten minutes at 72°C. PCR for rrs was conducted as follows 95°C for five minutes, 35 cycles of 95°C for one minute, 60°C for one minute, 72°C for one minute, and a final extension for seven minutes at 72°C. PCR for the rpoB was conducted as follows: 95°C for five minutes, 35 cycles of 95°C for one minute, 58°C for one minute, 72°C for one minute, and a final extension for seven minutes at 72°C. Amplified products were confirmed on a 1% Agarose gel stained with 4.6 ÎŒl SYBR safe DNA stain (Invitrogen, Carlsbad, California, USA), and results were visualized with an UltraViolet gel viewer (Terra Universal, S. Raymond Ave, Fullerton, CA, USA).\n\nThe PCR products were enzymatically purified using ExoSAP IT (Applied Biosystems, Foster City, California, USA). Purification conditions were done at 37°C for fifteen minutes followed by a second incubation at 80°C for fifteenminutes and a final cooling step at 4°C for five minutes.\n\nThe purified amplicons were sequenced in the forward and reverse directions by Sanger sequencing using Big Dye⢠Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) and the forward and reverse primers. The sequencing reaction for each gene was a 10 ÎŒl reaction comprising 1.25 ÎŒl of Big Dye Terminator, 3 ÎŒl of 5à Sequencing Buffer, 1 ÎŒl of 1 pmol of the sequencing primer, and 1.5 ÎŒl of the PCR product. The reaction volume was made up by adding 3.25 ÎŒl of nuclease-free water. The reaction proceeded through 96°C for 1 minute then 25 cycles of 96°C for 10 seconds, 50°C for five seconds, and 60°C for four minutes.\n\nPurification of cycle-sequencing products was done using the BigDye X Terminator⢠purification kit following the manufacturerâs instructions (Applied Biosystems, Foster City, California, USA) and purified products were loaded onto the ABI 3730 genetic analyzer (Applied Biosystems, Foster City, California, USA) for capillary electrophoresis.\n\nThe obtained sequences were first assembled into contigs and the consensus sequences aligned to their wild-type reference sequences for each gene using Geneious version 11.0 (Biomatters Ltd, Auckland, New Zealand). Mutations in the drug resistance genes were identified visually. STATA version 14 (StataCorp, College Station, Texas, USA) was used to test the association of NTM species to mutation patterns using Pearson chi-square at 95% confidence interval.\n\n\nResults\n\nOur study established that twenty-eight (23%) of NTM are resistant to at least one of the antibiotics in the macrolide-based therapy. One isolate (C47) containing M. abscessus had mutations conferring resistance in both rrl and rrs genes.\n\nThe bulk of drug-resistant isolates originated from the Lake Victoria, Coastal, and Nairobi regions, with six (5%) NTM showing mutations in the rrl, rrs, or rpoB genes (p=0.012). The age group was statistically significant (p=0.012), with isolates from participants aged 21 to 35 years old having the highest (n=10, 35.7 %) number of NTM with target gene alterations for rrl, rrs, and rpoB genes. Males accounted for twenty one (76.5%) of the 28 drug-resistant NTM identified, indicating a strong statistical significance (p=0.000). The majority of drug-resistant NTM isolates were detected in new (n=8; 28.5%) and TB relapse patients (n=8; 28.5%) patients, which had a significant statistical significance (p=0.001) (Table 2).\n\nMutations in rrl gene for the NTM were highly significant with a p value of <0.001. Twelve (10.4%) of NTM were resistant to macrolides (Table 2) with seven (58.3%) of RGM (Table 3) and five (41.7%) of SGM (Table 3) showing mutations within rrl gene. Point mutation at position 2058 was seen in M.intracellulare, M. abscessus subsp abscessus, M. nebraskense, M. massiliense, M. kumamototense, M. heraklionense, and M. bourgelatii. Only for M. abscessus subsp abscessus was a mutation at 2059 observed (Table 3).\n\nResistance to aminoglycosides was demonstrated by mutation at position A1408G of the rrs gene for six (11.1%) of the 54 RGM. The NTM with aminoglycoside resistance include M. abscessus subsp abscessus, M. chelonae, and M. alsense (Table 4). The NTM species had a low likelihood (p=0.06) of developing mutations in rrs gene for aminoglycoside resistance.\n\nA low association (p=0.89) for rifampicin resistance in SGM was observed. Mutations within codons between 503-533 of the rpoB were seen for ten (14.7%) SGM. These SGM included M. avium subsp avium, M. intracellulare, M. yongonense and M. gastri with mutations at codons 506, 509, 516, 526, and 531 respectively (Table 5).\n\n\nDiscussion\n\nNontuberculous mycobacteria (NTM) are an important cause of pulmonary disease worldwide, and are being isolated increasingly (Rivero-Lezcano & Carolina González-Cortés, 2019). They are often mistakenly treated as M. tuberculosis in countries devoid of laboratory competence for mycobacterial species differentiation (Pokam et al., 2022). Recently, there has been a considerable rise in infections caused by NTM (Saxena et al., 2021). These mycobacteria, which comprise a large and diverse range of species, have developed resistance to most conventional antibiotics, rendering their treatments unsatisfactory (Brown-Elliott et al., 2012).\n\nThis study established that 23% of NTM are resistant to at least one of the antibiotics in the macrolide-based therapy. Regional distribution of drug resistant NTM had a significant correlation (p=0.012) with the bulk of drug-resistant isolates originating from the Lake Victoria (n=6, 5%), Coastal (n=6, 5%), and Nairobi (n=6, 5%) regions. The observed regional diversity in drug resistant NTM across Kenya could be attributed to NTM evolution to evade natural antibiotics secreted as secondary metabolites by nearby environmental bacteria in various geographical landscapes (Moore et al., 2019). The 21-35 years age group had the highest number of NTM isolates presenting with drug resistance while males comprised the majority (n=21, 76.5%) of the drug-resistant NTM identified. NTM drug resistance was associated with a history of previous TB infection as seen in the high number of TB relapse cases recorded in this study (n=8; 28.5%, p=0.001). This is a common observation in sub-Saharan Africa given the high incidence of M. tuberculosis disease and the frequent misdiagnosis of NTM infection with TB seen in this region (Aliyu et al., 2013; Hoza et al., 2016; Pokam et al., 2022; Mwangi et al., 2022).\n\nDespite NTM demonstrating high levels of resistance to a broad range of antibiotics, macrolides including clarithromycin, erythromycin, and azithromycin remain the most effective antibiotic with >80% of isolates showing susceptibility (Ananta et al., 2018). However, some NTM including MAC and M. abscessus have been associated with increased resistance to macrolides leading to treatment failure (Saxena et al., 2021). Our study demonstrated a similar pattern where M. abscessus subsp abscessus (A2058G/C, A2059G) and M. intracellulare (A2058G) formed the majority of isolates (58.3%) with macrolide resistance. Other NTM presenting with high levels of resistance to macrolides were M. nebraskense (A2058C), M. massiliense (A2058T), M. kumamototense(A2058T), M. heraklionense (A2058T), and M. bourgelatii (A2058C). The increased potential for development of drug resistance in MAC species including M. intracellulare, and M. abscessus could be attributed to inherent factors such as a high propensity for genetic mutation in the drug target region, and high drug tolerance levels (Park et al., 2020), environmental factors facilitating the emergence of mutations in the rrl and subsequent ease of transmission to humans (Beverley Cherie Millar, 2019). Other mutations that could confer resistance to macrolides include T2419 in M. intracellulare (Huh et al., 2019). However, this mutation was not demonstrated in the Kenyan isolates of this study.\n\nThe commonly used aminoglycosides for the treatment of NTM are amikacin, streptomycin, kanamycin, tobramycin, and streptomycin (Krause et al., 2016). In our present study, three M. abscessus subsp abscessus, two M. chelonae, and one M. alsense presented with aminoglycoside resistance with A1408G mutation. Genotypic characteristics in rrs that indicate aminoglycoside resistance usually are in concordance with DST broth microdilution and GenoType NTM-DR assays, implying that mutations within rrs are the molecular basis of aminoglycoside resistance in NTM (Bouzinbi et al., 2020). For instance, a study that selected in-vitro aminoglycoside-resistant M. abscessus and M. chelonae presented an AâG mutation at position 1408 within the rrs upon sequencing. This confirms that a single point mutation at 1408 is adequate to confer high-level aminoglycoside resistance (Nessar et al., 2011). Further, individual mutations at T1406, C1409 and G1491 have also indicated considerable resistance to aminoglycoside in most M. abscessus subspecies (Nessar et al., 2011). Similar to other bacteria, NTM present with low-level aminoglycoside resistance through the production of drug-modifying enzymes including acetyltransferases (Sanz-GarcÃa et al., 2019), phosphorylases, adenylates, and methylases which act at various points on the aminoglycoside scaffold making it less potent (Krause et al., 2016; Munita & Arias, 2016; Sanz-GarcÃa et al., 2019; Tarashi et al., 2022; Zaragoza Bastida et al., 2017).\n\nSimilar to M. tuberculosis, resistance to rifampicin in NTM is mediated by mutations in the 81 bp RRDR corresponding to codons 503 to 533 of the rpoB gene (Saxena et al., 2021; Zhou et al., 2020). Our study identified ten (11.6%) rifampicin-resistant NTM with mutations occurring at codon 531 in M. avium, codon 506 in M. intracellulare, 509 in M. yongonense. M. gastri had amino acid substitutions at positions 516, 526, 531 of the rpoB gene. Our findings did not establish mutations outside of the RRDR. However, low-level rifampicin resistance has previously been demonstrated in M. intracellulare with mutations occurring outside the RRDR at position N494S (Park et al., 2020). Broth microdilution analysis which is the gold standard for rifampicin drug resistance testing presents a high minimum inhibition concentration (MIC) for isolates with mutations in the RRDR, hence confirming the role of these mutations in conferring high-level resistance to rifampicin (Huh et al., 2019). We further demonstrated that M. gastri harbored more than one mutation within the RRDR which is a unique attribute observed for M. kansasii complex species to which M.gastri belongs (Wu et al., 2018). A similar study obtained rifampicin-resistant M. kansasii from clinical isolates and in vitro generated mutant, and demonstrated mutations in codons 513, 526, and 531 of rpoB which is a common pattern in some SGM and M. tuberculosis (Klein et al., 2001).\n\nWe found considerable drug resistance in Kenyan NTM. To guide therapy, both species-level identification and drug resistance testing of NTM should be performed before starting treatment for NTM infection.\n\n\nConclusion\n\nWe demonstrated a significant level of drug resistance for macrolides, aminoglycosides and rifampicin in NTM isolated from symptomatic TB-negative patients in Kenya.\n\nM. abscessus and MAC were the dominant NTM with macrolide resistance, and most macrolide-resistant NTM harbored mutation at position 2058. Majority of RGM had mutation at position 1408 of the rrs gene, while rifampicin resistant SGM had mutations within the RRDR of the rpoB gene.\n\nThis study investigated the acquired mechanisms of drug resistance for NTM. Other intrinsic factors apart from drug target gene mutation could influence sensitivity of NTM to antibiotics.\n\nDespite this limitation, the study documents drug resistance mutation patterns of Kenyan NTM for the first time, and advocates for drug resistance testing before commencement of treatment for NTM infection.\n\nThis study was approved by Kenyatta National Hospital-University of Nairobi Ethics Review Committee (Ref: KNH-ERC/A/38) on 30th January 2020. Waiver for individual informed consent was granted as the study utilized remnant clinical samples and the research posed no greater than minimal risk to the study subjects.\n\n\nData availability\n\nFigshare. Mutation Patterns of Resistance Genes for Macrolides, Aminoglycosides, and Rifampicin in Nontuberculous Mycobacteria Isolates from Kenya. DOI: https://doi.org/10.6084/m9.figshare.20331378.v2.\n\nThis project contains the following underlying data:\n\n- Mutation patterns for rrl, rrs, and rpoB genes in NTM\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor contributions\n\nZMM - Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing â Original Draft Preparation, Writing â Review & Editing\n\nGN - Investigation, Writing â Review & Editing\n\nMWM - Supervision, Writing- Review and Editing\n\nFGO - Conceptualization, Methodology, Supervision, Writing- Review and Editing\n\nWDB - Conceptualization, Methodology, Supervision, Writing- Review and Editing",
"appendix": "Acknowledgments\n\nWe are grateful to the management of the National Public Health Laboratories- Kenya for granting us permission to access the National Tuberculosis Reference Laboratory and carry out this research. Much appreciation to the laboratory staff at the National Tuberculosis Reference.\n\nLaboratory for their technical support during the collection and initial analysis of sputum samples for this study.\n\n\nReferences\n\nAdékambi T, Colson P, Drancourt M: rpoB-Based Identification of Nonpigmented and Late-Pigmenting Rapidly Growing Mycobacteria. J. Clin. Microbiol. 2003; 41(12): 5699â5708. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlffenaar JW, MÀrtson AG, Heysell SK, et al.: Therapeutic Drug Monitoring in Non-Tuberculosis Mycobacteria Infections. Clin. Pharmacokinet. 2021; 60(6): 711â725. 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PubMed Abstract | Publisher Full Text\n\nBouzinbi N, Marcy O, Bertolotti T, et al.: Evaluation of the GenoType NTM-DR assay performance for the identification and molecular detection of antibiotic resistance in mycobacterium abscessus complex. PLoS One. 2020; 15(9 September 2020): 1â5. PubMed Abstract | Publisher Full Text\n\nBrown-Elliott BA, Iakhiaeva E, Griffith DE, et al.: In vitro activity of amikacin against isolates of mycobacterium avium complex with proposed MIC breakpoints and finding of a 16S rRNA gene mutation in treated isolates. J. Clin. Microbiol. 2013; 51(10): 3389â3394. PubMed Abstract | Publisher Full Text\n\nBrown-Elliott BA, Nash KA, Wallace RJ: Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clin. Microbiol. Rev. 2012; 25(3): 545â582. 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Microbiol. 2016; 34(4): 442â447. PubMed Abstract | Publisher Full Text\n\nHansen JL, Ippolito JA, Ban N, et al.: The structures of four macrolide antibiotics bound to the large ribosomal subunit. Structural Insights into Gene Expression and Protein Synthesis. 2002; 10: 117â128. Publisher Full Text\n\nHoza AS, Mfinanga SGM, Rodloff AC, et al.: Increased isolation of nontuberculous mycobacteria among TB suspects in Northeastern, Tanzania: public health and diagnostic implications for control programmes. BMC. Res. Notes. 2016; 9: 109. PubMed Abstract | Publisher Full Text\n\nHuh HJ, Kim SY, Jhun BW, et al.: Recent advances in molecular diagnostics and understanding mechanisms of drug resistance in nontuberculous mycobacterial diseases. Infect. Genet. Evol. 2019; 72(October 2018): 169â182. PubMed Abstract | Publisher Full Text\n\nKim SY, Kim DH, Moon SM, et al.: Association between 16S rRNA gene mutations and susceptibility to amikacin in Mycobacterium avium Complex and Mycobacterium abscessus clinical isolates. Sci. Rep. 2021; 11(1): 6108â6108. PubMed Abstract | Publisher Full Text\n\nKlein JL, Brown TJ, French GL: Rifampin resistance in Mycobacterium kansasii is associated with rpoB mutations. Antimicrob. Agents Chemother. 2001; 45(11): 3056â3058. PubMed Abstract | Publisher Full Text\n\nKoh W, Kwon OJ, Jeon K, et al.: Clinical Significance of Nontuberculous Mycobacteria Isolated From Respiratory Specimens in Korea. Chest. 2006; 129(2): 341â348. PubMed Abstract | Publisher Full Text\n\nKrause KM, Serio AW, Kane TR, Connolly LE: Aminoglycosides: An Overview.2016; 1â18.\n\nLi QJ, Jiao WW, Yin QQ, et al.: Compensatory mutations of rifampin resistance are associated with transmission of multidrug-resistant Mycobacterium tuberculosis Beijing genotype strains in China. 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Microbiol. 2017; 8(APR) Publisher Full Text\n\nNessar R, Reyrat JM, Murray A, et al.: Genetic analysis of new 16s rRNA mutations conferring aminoglycoside resistance in Mycobacterium abscessus. J. Antimicrob. Chemother. 2011; 66(8): 1719â1724. PubMed Abstract | Publisher Full Text\n\nOlivier KN, Griffith DE, Eagle G, et al.: Randomized trial of liposomal amikacin for inhalation in nontuberculous mycobacterial lung disease. Am. J. Respir. Crit. Care Med. 2017; 195(6): 814â823. PubMed Abstract | Publisher Full Text\n\nPark HE, Kim S, Shim S, et al.: 16S and 23S Rrna Gene Mutation Independent Multidrug Resistance of Non-Tuberculous Mycobacteria Isolated From South Korean Soil. Microorganisms. 2020; 8(8): 1â19. PubMed Abstract | Publisher Full Text\n\nPeixoto S, Maria L, Montenegro L, et al.: Major Article Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting. Journal of the Brazilian Society of Tropical Medicine. 2020; 53: 1â7. Publisher Full Text\n\nPharmd JAS, Escalante P, Wilson JW: Pharmacotherapy Approaches in Nontuberculous Mycobacteria Infections Jennifer. Mayo Clin. Proc. 2019; 94(August): 1567â1581. PubMed Abstract | Publisher Full Text\n\nPokam BDT, Yeboah-Manu D, Ofori S, et al.: Prevalence of non-tuberculous mycobacteria among previously treated TB patients in the Gulf of Guinea, Africa. IJID Regions. 2022; 3(January): 287â292. PubMed Abstract | Publisher Full Text\n\nRamasoota P, Pitaksajjakul P, Phatihattakorn W, et al.: Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains from Thailand and its evolutionary implication. Southeast Asian J. Trop. Med. Public Health. 2006; 37(1): 136â147. PubMed Abstract\n\nRivero-Lezcano OM, Carolina González-Cortés MM: The Unexplained Increase of Nontuberculous Mycobacteriosis Octavio. Int. J. Mycobacteriol. 2019; 8(1): 1â6. PubMed Abstract | Publisher Full Text\n\nSam AS, Ninan MM, Ranjani R, et al.: Nontuberculous mycobacteria â clinical and laboratory diagnosis: experiences from a TB endemic country. Future Science OA. 2020; 6. Publisher Full Text\n\nSanz-GarcÃa F, Anoz-Carbonell E, Pérez-Herrán E, et al.: Mycobacterial aminoglycoside acetyltransferases: A little of drug resistance, and a lot of other roles. Front. Microbiol. 2019; 10(JAN): 1â11. PubMed Abstract | Publisher Full Text\n\nSaxena S, Spaink HP, Forn-Cunà G: Drug resistance in nontuberculous mycobacteria: Mechanisms and models. Biology. 2021; 10(2): 1â22. PubMed Abstract | Publisher Full Text\n\nSimons S, Van Ingen J , Hsueh P, et al.: Nontuberculous Mycobacteria in Respiratory Tract Infections, Eastern Asia. Emerg. Infect. Dis. 2011; 17(3). Publisher Full Text\n\nTarashi S, Siadat SD, Fateh A: Nontuberculous Mycobacterial Resistance to Antibiotics and Disinfectants: Challenges Still Ahead. Biomed. Res. Int. 2022; 2022: 1â12. PubMed Abstract | Publisher Full Text\n\nWHO: WHO guidelines for the programmatic management of drug-resistant tuberculosis.2014; (pp. 1â430).Reference Source\n\nWu ML, Aziz DB, Dartois V, et al.: NTM drug discovery: status, gaps and the way forward. Drug Discov. Today. 2018; 23(8): 1502â1519. PubMed Abstract | Publisher Full Text\n\nZaragoza Bastida A, Rivero Pérez N, Valladares Carranza B, et al.: Molecular Identification of Mycobacterium Species of Public Health and Veterinary Importance from Cattle in the South State of México. Canadian Journal of Infectious Diseases and Medical Microbiology. 2017; 2017: 4â11. PubMed Abstract | Publisher Full Text\n\nZhou L, Xu D, Liu H, et al.: Trends in the Prevalence and Antibiotic Resistance of Non-tuberculous Mycobacteria in Mainland China, 2000â2019: Systematic Review and Meta-Analysis. Front. Public Health. 2020; 8(July): 1â9. Publisher Full Text"
}
|
[
{
"id": "148288",
"date": "22 Sep 2022",
"name": "Leena Al-Hassan",
"expertise": [
"Reviewer Expertise Infectious diseases and antibiotic resistance"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study characterises mutation patterns for resistance genes in NTM from Kenya.\n\nAlthough the study is interesting, I think the authors need to give more information in the methodology and results section on:\nIdentification: 122 isolates were collected, but no details in the results on how many were slow-growing and rapid-growing NTM.\n\nMIC: No mention of methodology for MIC and results.\n\nThe PCR: Did you include any positive or negative controls in the experiments?\n\nThe discussion on mutations and their role in resistance is quite vague. It's not clear whether the mutations identified can be linked to the observed MIC. Have the researchers checked that these are not silent mutations?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "8825",
"date": "06 Oct 2022",
"name": "Zakayo Mwangi",
"role": "Author Response",
"response": "I appreciate your review. Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.  The phenotypic drug sensitivity by MIC analysis was not done in this study. We performed genotypic characterization of the drug target genes by describing the mutations associated with drug resistance through target sequencing of the drug target genes.  Positive and negative controls were included during PCR and subsequent sequencing of respective target genes. The controls were based on Genotype NTM-DR results.  In the discussion, the mutations identified in this study have been compared to those seen in previous studies that had also included MIC in their analysis. The point mutations identified in the drug target genes were seen to be associated with phenotypic drug resistance."
}
]
},
{
"id": "150334",
"date": "10 Oct 2022",
"name": "Utpal Sengupta",
"expertise": [
"Reviewer Expertise Infectious diseases with special reference to tuberculosis and leprosy",
"Cell Biology",
"Microbiology",
"Immunology and Biotechnology."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript has been written on the findings of antimycobacterial resistance of NTM isolated from 122 sputum M. tuberculosis-negative patients. The authors have investigated their resistance patterns on the isolated strains using gene sequencing of the drug-resistant genes and noted the mutations in the respective codon positions.\nMaterials and methods have been well described. Data were properly analyzed and presented adequately in the tables. However, there were a lot of discrepancies in Table 2 in the first column where the total sample number was shown to be 135 instead of 122 and therefore the percentage expressions were wrongly mentioned in the table and in the text. The percentages have to be calculated correctly and Table 2 has to be modified and described accordingly in the text.\nThe discussion has been adequate, citing adequate references. However, the discussion with respect to the results of Table 2 has to be modified and discussed for a correct understanding of the reader.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "8910",
"date": "24 Oct 2022",
"name": "Zakayo Mwangi",
"role": "Author Response",
"response": "I appreciate your review. The issue mentioned above has been addressed."
}
]
}
] | 1
|
https://f1000research.com/articles/11-962
|
https://f1000research.com/articles/12-126/v1
|
02 Feb 23
|
{
"type": "Research Article",
"title": "Predicting infection with COVID-19 disease using logistic regression model in Karak City, Jordan",
"authors": [
"Anas Khaleel",
"Wael Abu Dayyih",
"Lina AlTamimi",
"Liana Dalaeen",
"Zainab Zakaraya",
"Alhareth Ahmad",
"Baker Albadareen",
"Abdallah Ahmed Elbakkoush",
"Anas Khaleel",
"Wael Abu Dayyih",
"Lina AlTamimi",
"Liana Dalaeen",
"Zainab Zakaraya",
"Alhareth Ahmad",
"Baker Albadareen"
],
"abstract": "Background: On March 2020, World Health Organization (WHO) labeled coronavirus disease 2019 (COVID-19) as a pandemic. COVID-19 has rapidly increased in Jordan which resulted in the announcement of the emergency state on March 19th, 2020. Despite the variety of research being reported, there is no agreement on the variables that predict COVID-19 infection. We have analyzed the data collected from Karak city citizens to predict the probability of infection with COVID-19 using binary logistic regression model. Methods: Based on data collected by Google sheet of COVID-19 infected and non-infected persons in Karak city, analysis was applied to predict COVID-19 infection probability using a binary logistic regression model. Results: The ultimate logistic regression model provides the formula of COVID-19 infection probability based on sex and age variables. Conclusions: Given a person's age and sex, the final model presented in this study can be used to calculate the probability of infection with COVID-19 in Karak city. This could help aid health-care management and policymakers in properly planning and allocating health-care resources.",
"keywords": [
"COVID-19",
"Google Sheet",
"Logistic regression model",
"Sex",
"Age",
"Smoking"
],
"content": "Introduction\n\nIn December 2019, coronavirus disease 2019 (COVID-19) was first reported in Wuhan city, China.1,2 It wasn't long before it was determined that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19.3 This virus spread quickly all over the world and was declared by the World Health Organization as a pandemic.4\n\nMost of the studies have concentrated on the outbreak in China since the first reported cases were published, including the disease's transmission, risk factors for infection, and biological features of the virus using different statistical models as can be seen in literatures.5â9 An exponential model was used to predict the number of infected people in Italy based on the data reported by the Italian Health Ministry.10 Maleki et al examined the data of confirmed and recovered COVID-19 cases using a set of two-piece scale mixture normal distributions models.11\n\nThe Susceptible-Infective-Recovered (SIR) model was used to anticipate the characteristics of COVID-19 cases in China.12 Caccavo et al proposed a modified Susceptible, Infected, Removed, and Dead (SIRD) model to estimate how the COVID-19 outbreaks in China and Italy will develop.13 In another work a deep-learning algorithm called long short-term memory (LSTM) was used to anticipate COVID-19 cases in Iran.14\n\nThe first diagnosed COVID-19 case in Jordan was reported in March 2020, a Jordanian citizen who had returned from Italy.15 At the end of 2020 Jordan reported more than 271,000 COVID-19 verified cases and more than 3,500 fatalities.16\n\nIn this work we used a binary regression model to fit the data collected from Karak city citizens by Google sheet. We were able to build a final equation that can predict the probability of infection with COVID-19 in Karak city based on sex and age variables.\n\n\nMethods\n\nThis study was approved by ethical committee in Mutah University and University of Petra (MUTAH-UOP no.:20219091) on 9th September 2021. Informed consent was obtained from the study subjects, via a question at the start of the survey: âI agree to answer questions: yes/noâ. Those who refused to answer or did not want to continue answering the questions were allowed to opt out any time. The ethical approval number was posted on the top of questionnaire first page and an email and telephone number of the principal investigator was also posted in case of any question or inquiry.\n\nA structured questionnaire via google sheet was used to gather information from Karak city residents. The information was collected from September 10th to the end of October 2021. The survey has employed a variety of demographic variables including sex, age, job, smoking, chronic disease, yearly flu injection, and infected with COVID-19 before vaccination. All participants received an explanation of the questionnaire's goals and objectives at the outset of the survey.\n\nThe questionnaire was written by the authors in Arabic language, translated two ways and provided as Extended data.26 The questionnaires' reliability and validity were not performed, and the authors couldnât follow up the participants since they didnât provide their personal contact information.\n\nIn order to avoid any potential bias in our study the survey questions were clear, direct, and the sequence order of the questions were designed in a way to avoid influencing the participantsâ answers. Moreover, we didnât limit the time period for the participants to complete the questionnaire and the selection of participants was random.\n\nIn this work the Raosoft sample size calculator was used to determine the expected sample size.17 Based on a 50% response rate, a 95% confidence interval, and a 5% margin of error, the sample size was estimated. The maximum sample size needed is 377. Consequently, this research used a practical sample of 386 persons out of 402 participants. A total of 16 participants were excluded from the study due to missing data. Being an adult (older than 18 years old) and residing in Karak city during the study were requirements for inclusion.\n\nThe research method employed was a cross-sectional study design with random convenience sampling. Using Google Forms, an anonymous survey was posted online and shared on popular social media sites including Facebook and WhatsApp.\n\nThe IBM SPSS statistics 22 software was used to evaluate the data. The binary logistic regression model and the likelihood ratio (LR) chi-square test were used in the analysis. A significant P-value < 0.05 was statistically considered.\n\n\nResults\n\nA total of 323 of the 386 participants that responded to the survey were women, while 63 of the participants were men.25 In this study there were two categories for age: one related to participants aged less than or equal 45 years old (<=45) and the other one related to participants aged over 45 years old (>45). This age cut value has been chosen based on a study was done in United States in 2020. The results of this study showed that the number of infected people with COVID-19 was higher among those aged 40 â 49 years old.18 In our study we have calculated the median age of that interval which results in the age cut value 45 years old.\n\nThe statistical analysis of the collected data for different geographic variables shows that 295 (76.4%) participants were aged less than or equal 45 years old, while 91 (23.6%) persons were aged over 45 years old. The number of participants who had non-medical job was found to be 166 (43%) from the whole participants, while 77 (19.9%) are working in the medical field, in addition to 69 (17.9%) students, and finally a total of 74 (19.2%) are unemployed.\n\nMoreover, the number of participants who smoke in our sample is 68 (17.6%), the former smokers were 317 (82.1%), and 1 participant was a non-smoker. In total, 91 (24.6%) participants had a chronic disease, and a total of 291 (75.4%) persons didnât suffer from any disease. The number of the persons who took the yearly flu vaccine were found to be 40 (10.4%) persons, and 346 (89.6%) didnât take the yearly flu vaccine. All demographic variables of the participants in this study are shown below in Table 1.\n\nThe statistical analysis of our sample shows that 257 of the participants have been infected with COVID-19 and they are distributed with respect to demographics variables as shown in Table 2 below.\n\nTable 2, shows that among the 275 persons infected with COVID-19, 223 (86.8%) were women, 167 of them aged <= 45 years old and 56 of them aged over 45 years old. For the 34 (13.2%) infected men 21 of them were aged <= 45 years old while 13 were aged over 45 years old. This leads to a total of 188 (73.2%) of the infected participants aged <= 45 years old, and 69 (26.8%) aged over 45 years old.\n\nThe number of infected participants working in the medical field was 52 (20.2%) (45 women and 7 men). Among the infected there were 120 (46.7%) participants with a non-medical job (101 women and 19 men), 41 (16%) were infected students (35 women and 6 men), and 44 (17.5%) were unemployed infected participants (42 women and 2 men). Furthermore, the total number of infected participants who are former smokers were 211 (82.1%) (199 women and 12 men), while 45 (17.5%) (23 women and 22 men) were infected smokers.\n\nOverall, 60 (23.3%) (51 women and 9 men) were infected participants with a chronic disease, 197 (76.7%) (172 women and 25 men) were infected without any disease. Moreover, 26 (10.1%) (18 women and 8 men) participants were flu vaccinated and infected with COVID-19, while 231 (89.9%) (205 women and 26 men) was infected with COVID-19 and didnât take flu vaccine.\n\nAll the demographic variables in this study were tested using LR chi-square test to determine the probability of COVID-19 infection predictors. The results show that sex and age of the participants are the significant demographic infection predictors. Table 3 provides counts and ratios between these predictor variables.\n\nAmong the 295 participants aged <= 45 years old 252 (78%) were women and 43 (68.3%) of them were men. For the 91 participants who aged > 45 years old 71 (22%) of them were women and 20 (31.7%) were men.\n\nIn our binary logistical regression model, we have used all predictor variables (sex and age). The results of the model are shown below in Table 4.\n\nCoefficients values of the model and their statistical significance P-value were obtained by âEnter logistical regression methodâ. LR chi-square test was applied to evaluate the overall model fit and to test the significant coefficients. The B coefficient values were found to be (-0.716, 0.48), while the Wald Statistics values are (6.307, 5.438) for sex and age respectively. The exponentiated logistic coefficient (Exp (B)) shows the values of 0.489 for sex and 1.911 for age cut value 45 years old.\n\nOur model in this work uses two indicators to measure model fineness percentage; Cox & Snell R Square (R2 = 0.028) and Nagelkerke R Square (R2 = 0.039). Although the R2 values are too small, it indicated a weak relationship. This value explanted that out model contributes about 4% of the COVID-19 infection probability as illustrated in Table 5 below. It is important to mention that the Cox & Snell R Square indicator commonly produced underestimates the real value.19,20\n\nThis research aimed to predict the probability of infection with COVID-19 in Karak city. Using the final logistic regression model data presented in Table 4 results, the formula of COVID-19 infection probability (Pinfected) is given as:\n\nThe probability of infection can be calculated by substituting the values for sex and age in equation (1).\n\n\nDiscussion\n\nAccording to the results obtained from Table 2 the number of infected women with COVID-19 was 223 (86.8%) and the number of infected men was 34 (13.2%). This difference is in agreement with the results of a study in the United States recorded from January to May 2020, where the number of infected women with COVID-19 was 51.1%, while the number of infected males was 48.9%.18\n\nIn another study, infection rates for COVID-19 were reconstructed by age and sex using data from different European countries.21 The results show in all the analyzed countries, the chance of infection with COVID-19 among women increases more sharply after age 20 until late 50s.21\n\nThese results support the ones obtained in our study since among the 188 (73.2%) participants aged <= 45 years infected with COVID-19, 167 (88.8%) of them were women and 21 (11.2%) were males.\n\nMoreover, in this work among the former and current smokers infected with COVID-19, 222 (86.7%) (199 former and 23 current) are women, while 34 (13.3%) (12 former and 22 current) are men. These results are consistent with previous studies which recognized that one of the risk factors associated with COVID-19 is smoking.22â24\n\nThe results of the exponentiated logistic coefficient show that the probability of women to be infected with COVID-19 is more than men for the same age. This result is in agreement with the results obtained from our model (equation (1)) for calculating the probability of infection with COVID-19.\n\nIn equation (1) the sex variable labeled by (0) for women, (1) for men, while the age variable labeled by (0) if age less than or equal 45 years old and labeled (1) if age is more than 45 years old. Using equation (1) we can calculate the probability of a man aged 33 years old to be infected with COVID-19 by substituting numbers for age and sex. The results lead to Pinfected = 0.4895. Since Pinfected is less than 0.5, indicates the man is not infected. Repeating the same calculations for a woman with the same age result in Pinfected = 0.6624, indicates the woman is infected with COVID-19.\n\nA notable limitation of this study was its brief lifespan. There was no more observation to validate the model. Furthermore, we are unable to extrapolate our results to the other cities in Jordan because we only evaluated people in Karak city.\n\n\nConclusion\n\nGiven a person's age and sex, equation (1) presented in this study can be used to calculate the probability of infection with COVID-19 in Karak city. This statistical model can be used to forecast outbreak trends. This forecast could aid health-care management and policymakers in properly planning and allocating health-care resources.",
"appendix": "Data availability\n\nFighsare: supplementary data.xlsx. https://doi.org/10.6084/m9.figshare.21829650. 25\n\nFigshare: questionnaire supplementary information.docx https://doi.org/10.6084/m9.figshare.21931731. 26\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe would like to offer our heartfelt gratitude to the Faculty of Pharmacy and Medical Sciences at the University of Petra and Mutah University in Jordan, for providing us with the chance to successfully complete this research. The authors would also like to thank the Deanship of Scientific Research and Graduate Studies for their invaluable assistance. Furthermore, we would like to acknowledge the role of Mohammad Niazi in the process of collecting data and advertising the questionnaire.\n\n\nReferences\n\nLescure FX, Bouadma L, Nguyen D, et al.: Clinical and virological data of the first cases of COVID-19 in Europe: a case series. Lancet Infect. Dis. 2020; 20(6): 697â706. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497â506. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKsiazek TG, Erdman D, Goldsmith CS, et al.: A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 2003; 348(20): 1953â1966. Publisher Full Text\n\nWHO: Coronavirus disease 2019 (COVID-19): Situation report 111. 2020.\n\nWu JT, Leung K, Bushman M, et al.: Estimating clinical severity of COVID-19 from the transmission dynamics in Wuhan, China. Nat. Med. 2020; 26(4): 506â510. Publisher Full Text\n\nLi Q, Guan X, Wu P, et al.: Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia. N. Engl. J. Med. 2020; 382(13): 1199â1207. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu JT, Leung K, Leung GM: Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet. 2020; 395(10225): 689â697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu N, Zhang D, Wang W, et al.: A Novel Coronavirus from Patients with Pneumonia in China, 2019. N. Engl. J. Med. 2020; 382(8): 727â733. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao S, Lin Q, Ran J, et al.: Preliminary estimation of the basic reproduction number of novel coronavirus (2019-nCoV) in China, from 2019 to 2020: A data-driven analysis in the early phase of the outbreak. Int. J. Infect. Dis. 2020; 92: 214â217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRemuzzi A, Remuzzi G: COVID-19 and Italy: what next? Lancet. 2020; 395(10231): 1225â1228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaleki M, Mahmoudi MR, Wraith D, et al.: Time series modelling to forecast the confirmed and recovered cases of COVID-19. Travel Med. Infect. Dis. 2020; 37: 101742. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNesteruk I: Statistics-based predictions of coronavirus epidemic spreading in mainland China. 2020.\n\nCaccavo D: Chinese and Italian COVID-19 outbreaks can be correctly described by a modified SIRD model. MedRxiv. 2020.\n\nAyyoubzadeh SM, Ayyoubzadeh SM, Zahedi H, et al.: Predicting COVID-19 incidence through analysis of google trends data in iran: data mining and deep learning pilot study. JMIR Public Health Surveill. 2020; 6(2): e18828. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAl-Balas M, Al-Balas HI, Jaber HM, et al.: Distance learning in clinical medical education amid COVID-19 pandemic in Jordan: current situation, challenges, and perspectives. BMC Med. Educ. 2020; 20(1): 1â7.\n\nEl-Elimat T, AbuAlSamen MM, Almomani BA, et al.: Acceptance and attitudes toward COVID-19 vaccines: A cross-sectional study from Jordan. PLoS One. 2021; 16(4): e0250555. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaosoft I: Raosoft sample size calculator. 2004 [cited 2021 July 13].Reference Source\n\nStokes EK, Zambrano LD, Anderson KN, et al.: Coronavirus Disease 2019 Case Surveillance - United States, January 22-May 30, 2020. MMWR Morb. Mortal. Wkly Rep. 2020; 69(24): 759â765. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZewude BT, Ashine KM: Binary Logistic Regression Analysis in Assessment and Identifying Factors That Influence Students' Academic Achievement: The Case of College of Natural and Computational Science, Wolaita Sodo University, Ethiopia. J. Educ. Pract. 2016; 7(25): 3â7.\n\nDe La Viña L, Ford J: Logistic Regression Analysis of Cruise Vacation Market Potential: Demographic and Trip Attribute Perception Factors. J. Travel Res. 2001; 39(4): 406â410. Publisher Full Text\n\nSobotka T, Brzozowska Z, Muttarak R, et al.: Age, gender and COVID-19 infections. MedRxiv. 2020.\n\nLiu W, Tao ZW, Wang L, et al.: Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease. Chin. Med. J. 2020; 133(9): 1032â1038. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang JJ, Dong X, Cao YY, et al.: Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan, China. Allergy. 2020; 75(7): 1730â1741. PubMed Abstract | Publisher Full Text\n\nGuan WJ, Ni ZY, Hu Y, et al.: Clinical Characteristics of Coronavirus Disease 2019 in China. N. Engl. J. Med. 2020; 382(18): 1708â1720. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElbakkoush A: supplementary data.xlsx. Dataset. figshare. 2023. Publisher Full Text\n\nElbakkoush A:survey supplementary information.docx. Dataset. figshare. 2023. Publisher Full Text"
}
|
[
{
"id": "162334",
"date": "08 Feb 2023",
"name": "Muna Barakat",
"expertise": [
"Reviewer Expertise Pharmacy practice and public health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for inviting me to review this work. This manuscript entitled â Predicting infection with COVID-19 disease using logistic regression model in Karak City, Jordanâ aimed to test the predictors that probably contributed to the infection with COVID-19 using a binary logistic regression model. I believe this study is important at the local and international levels. Hence, the evidence is not well established in the literature.\nMinor comments must be addressed before publication:\nAbstract:\nPlease remove the statement, âWe have analyzed the data collected from Karak city citizens to predict the probability of infection with COVID-19 using binary logistic regression model.â And re-write the aim as the following instead âThis study aimed to test the predictors that probably contributed to the infection with COVID-19 using a binary logistic regression model.â\n\nResults of the abstract:\nPlease add the total number of participants, demographics, and main outcomes of the logistic regression (Odds ratio and p-value).\n\nIntroduction:\nPlease elaborate more in the introduction, especially in the context of Jordan. There are loads of Jordanian studies within the era of COVID-19. End the introduction with the aim and remove âIn this work we used a binary regression model to fit the data collected from Karak city citizens by Google sheet. We were able to build a final equation that can predict the probability of infection with COVID-19 in Karak city based on sex and age variables.â\n\nMethods:\nPlease add a section for tool development (with citation) and validation. Please indicate if you have checked the regression- assumptions in the data analysis section. Any validation for the equation?\n\nDiscussion:\nI believe it is too short, you need to discuss/interpret the results thoroughly to show/highlight the importance of the study findings. Please elaborate more.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9387",
"date": "27 Feb 2023",
"name": "abdallah elbakkoush",
"role": "Author Response",
"response": "Reply to Reviewer comments Abstract: please remove the statement, âWe have analyzed the data collected from Karak city citizens to predict the probability of infection with COVID-19 using binary logistic regression model.â And re-write the aim as the following if suit âThis study aimed to test the predictors that probably contributed to the infection with COVID-19 using a binary logistic regression model.â A: We thank the reviewer for the suggestion of rewriting the statements. The sentence in the aim of the study was re-written. Results of the abstract: please add the total number of participants, demographics, and main outcomes of the logistic regression (Odds ratio and p-value). A: We thank the reviewer for pointing this out. We have added the required information in the results of the abstract. A total of 386 participants have completed the questionnaire including 323 women and 63 men. Among the participants 295 (76.4%) were aged less than or equal 45 years old, and 91 (23.6%) were aged over 45 years old. Of all participants, 166 (43%) had non-medical jobs; meanwhile, 77 (19.9%) were employed in the medical area; additionally, 69 (17.9%) were students; and finally, 74 (19.2%) were unemployed. In addition, 68 (17.6%) of the people in our sample smoke, 317 (82.1%) were former smokers, and 1 person did not smoke. A total of 291 participants (75.4%) were disease-free, whereas 91 (24.6%) participants had a chronic illness. 40 people (10.4%) were determined to have received the annual flu shot; 346 people (89.6%) did not receive the annual flu shot. Among the 386 participants a total of 275 were infected with COVID-19. The LR chi-square test was used to analyze every demographic characteristic in this study to find predictors of the likelihood of COVID-19 infection. The findings indicate that the participants' sex and age are the most important demographic determinants of infection. Cox & Snell R Square (R2 = 0.028) and Nagelkerke R Square (R2 = 0.039) indicators was used to measure model fineness with significant P-value < 0.05. Introduction: Please elaborate more in the introduction, especially in the context of Jordan. There are loads of Jordanian studies within the era of COVID-19. A: We thanks for the reviewer for pointing out this. We modified the introduction and included more studies regarding COVID-19 in Jordan. The Covid-19 epidemic inspired numerous investigations in Jordan. For instance, health care professionals, particularly pharmacists, believed that they required training in psychological care in order to support people during local pandemics as well as themselves (https://doi:10.1186/s40545-020-00254-y ).  The majority of Jordan's general public recognized how the covid disease was spread and that chronic illnesses like diabetes and aging were risk factors as well as being immunocompromised (https://doi:10.31838/srp.2020.7.22). Odeh et al developed a prediction model of risk factors for complications among COVID-19 patients in Jordan. The results of the model showed that diabetes, shortness of breath, Body Mass Index (BMI), and abnormal neutrophils are the risk factors associated with COVID-19 complications (https://DOI:10.1016/j.jiph.2021.02.010). In another study, climate indicators including average daily temperature, relative humidity, wind velocity, pressure, and concentrations of pollutants were used to predict COVID-19 active cases in the three cities in Jordan using Artificial Neural Networks (ANN) and multiple linear regression (https://doi:10.1007/s12665-022-10348-2). Moreover, Hussein et al proposed three approaches (short-term forecast (STF) model, long-term forecast (LTF) model, and hybrid forecast (HF) model) for predicting COVID-19 pandemic's development in Jordan over a long-term period that took into account various social circumstances like everyday life, curfews, lockdowns, and vaccination (https://doi:10.3390/vaccines9070728). End the introduction with the aim and we remove âIn this work we used a binary regression model to fit the data collected from Karak city citizens by Google sheet. We were able to build a final equation that can predict the probability of infection with COVID-19 in Karak city based on sex and age variables.â A: We thank the reviewer for the comments. We have removed these statements and added the aim of the study at the end of the introduction. âWe were able to build a final equation that can predict the probability of infection with COVID-19 in Karak city based on sex and age variables.â  Methods: Please add a section for tool development (with citation) and validation. A: We thank the reviewer for the suggestion. Tool development section was added to the methods and we have included that the questionnaires' reliability and validity were not performed. Please indicate if you have checked the regression- assumptions in the data analysis section. Any validation for the equation? A: We the reviewer for pointing this out. All logistic regression assumptions were checked, and all are valid for our model. This statement was added at the end of the data analysis section: âAll of the Essential Assumptions for Implementing Logistic Regression model were evaluated and determined to be validâ. Any validation for the equation? A random validation of the equation was tested throughout the study. She was a random female over the age of 45 years old who had contracted COVID-19 again.  Discussion: I believe it is too short, you need to discuss/interpret the results thoroughly to show/highlight the importance of the study findings. Please elaborate more. A: We thank the reviewer for pointing out this. The following paragraphs was added in the discussion section.  Furthermore, in our sample 197 participants are suffering from chronic disease and have been infected with COVID-19. Previous study found that the increased COVID-19 severity and increased admittance to intensive care unit (ICU) were strongly correlated to preexisting chronic diseases (http://dx.doi.org/10.14336/AD.2020.0502). Another study found that people with obesity, cardiovascular disease, hypertension, and neuromuscular illness were much more likely to experience severe pandemic COVID-19 (https://doi.org/10.1136/bmj.f5061). Moreover, Laires et al revealed that renal, cardiovascular, and respiratory diseases were associated with ICU admission and mortality within COVID-19 infected patients (The Association Between Chronic Disease and Serious COVID-19 Outcomes and Its Influence on Risk Perception: Survey Study and Database Analysis. JMIR Public Health Surveill. 2021 Jan 12;7(1):e22794. doi: 10.2196/22794. PMID: 33433397; PMCID: PMC7806339). Among the 257 COVID-19 infected participants, 231 of them didnât receive the yearly flu vaccine. Recent existing data in literatures indicates that influenza vaccination may lessen the COVID-19 clinical consequences. A meta-analysis study revealed a significant advantage for mechanical ventilation in COVID-19 individuals who had received an influenza vaccination over those who had not (https://doi.org/10.1038/s41598-022-18618-6 ). Another meta-analysis of observational studies included 290,327 participants claimed that receiving an influenza vaccination decreases the chance of COVID-19 infection which is in agreement with the findings of our study (https://doi.org/10.3390/vaccines9050529 ). On the other hand, the relationship between the influenza vaccine and a decreased risk of COVID-19 negative outcomes was discussed by Fink et al (https://doi.org/10.1136/bmjebm-2020-111549 ). This included the ability of vaccines to activate innate immunity and produce known \"off-target\" protection against infections other than those specifically targeted by the vaccine (https://doi.org/10.1136/bmjebm-2020-111549 )."
}
]
}
] | 1
|
https://f1000research.com/articles/12-126
|
https://f1000research.com/articles/11-1323/v1
|
15 Nov 22
|
{
"type": "Review",
"title": "Different levels of circadian (de)synchrony Ââ where does it hurt?",
"authors": [
"Ankita AS. Galinde",
"Faheem Al-Mughales",
"Henrik Oster",
"Isabel Heyde",
"Ankita AS. Galinde",
"Faheem Al-Mughales",
"Henrik Oster"
],
"abstract": "A network of cellular timers ensures the maintenance of homeostasis by temporal modulation of physiological processes across the day. These so-called circadian clocks are synchronized to geophysical time by external time cues (or zeitgebers). In modern societies, natural environmental cycles are disrupted by artificial lighting, around-the-clock availability of food or shiftwork. Such contradictory zeitgeber input promotes chronodisruption, i.e., the perturbation of internal circadian rhythms, resulting in adverse health outcomes. While this phenomenon is well described, it is still poorly understood at which level of organization perturbed rhythms impact on health and wellbeing. In this review, we discuss different levels of chronodisruption and what is known about their health effects. We summarize the results of disrupted phase coherence between external and internal time vs. misalignment of tissue clocks amongst each other, i.e., internal desynchrony. Last, phase incoherence can also occur at the tissue level itself. Here, alterations in phase coordination can emerge between cellular clocks of the same tissue or between different clock genes within the single cell. A better understanding of the mechanisms of circadian misalignment and its effects on physiology will help to find effective tools to prevent or treat disorders arising from modern-day chronodisruptive environments.",
"keywords": [
"Chronodisruption",
"circadian misalignment",
"coupling",
"inter-tissue desynchrony",
"intra-tissue desynchrony",
"phase incoherence",
"shiftwork"
],
"content": "Introduction\n\nMammals possess a ubiquitously expressed circadian clock system with a master pacemaker located in suprachiasmatic nucleus (SCN), in the hypothalamus, driving physiological and behavioral rhythms. Such rhythms can be observed in, e.g., hormonal release, eating patterns, sleep behavior and body temperature (Moore and Eichler, 1972; Ralph et al., 1990; Sawaki et al., 1984; Stephan and Zucker, 1972). Functional clocks have been found in the SCN but also numerous other tissues including liver, kidney, and adipose tissues (Aschoff, 1965; Aschoff et al., 1967; Balsalobre et al., 1998; Lamia et al., 2008; Yamazaki et al., 2000; Yoo et al., 2004). All circadian clocks share three common properties. First, circadian oscillators are self-sustained, i.e., they are capable of driving ~24-hour circadian rhythms in transcription and translation. Second, they preserve the same kinetics over a broad range of temperatures (Barrett and Takahashi, 1995; Menaker and Wisner, 1983; Pittendrigh, 1954; Reyes et al., 2008). Third, circadian oscillators can be synchronized (or entrained) by environmental cues, so called zeitgebers (German for âtime giverâ) such as light and food (Balsalobre et al., 1998; Yoo et al., 2004). Photic zeitgeber input synchronizes the SCN that in turn aligns the central nervous system (CNS) and peripheral-tissue clocks with each other and with external time via humoral and neural signals. Together, these give rise to rhythmic circadian output (Figure 1). Single circadian oscillators have distinct period lengths, and it is thought that they must be synchronized to the 24-hour light/dark (LD) cycle to provide coherent rhythmic control over physiological processes such as maintaining temporal separation of chemically incompatible processes (Honma et al., 1998; Liu et al., 2007; Nagoshi et al., 2004; Welsh et al., 1995). Modern lifestyles are often characterized by deregulated food intake rhythms, lack of exercise, disrupted sleep/wake patterns, and nocturnal light exposure. Such mismatch of zeitgeber signals is believed to lead to disruptions in the phase coherence between internal and external time and between different tissue clocks in a state termed chronodisruption (Erren and Reiter, 2009, 2013; Kiehn et al., 2017).\n\nThe primary zeitgeber, light, entrains the master pacemaker, the suprachiasmatic nucleus (SCN), with geophysical time. The SCN synchronizes subordinated central nervous system (CNS) and peripheral tissue clocks by humoral and neural signals and the temporal coordination of food intake, body temperature, and rest/activity cycles. Outputs of peripheral tissues feedback to clocks in the brain and stabilize circadian synchrony. Integration of external signals, collective output of tissue clocks, and rhythmic humoral and neural signals generates physiological and behavioral circadian rhythms. Figure created with BioRender.com.\n\nThe circadian clock system regulates energy homeostasis, and dysregulation of circadian clock-metabolism crosstalk can seriously impact overall metabolic health (Reinke and Asher, 2019). For example, eating at a biologically inappropriate phase results in impaired glucose tolerance and transient insulin resistance in a laboratory study (Scheer et al., 2009). Nighttime eaters gain more weight compared to non-nighttime eaters (Gluck et al., 2008). Increased light at night (LAN) exposure is associated with a higher incidence of obesity and metabolic syndrome (McFadden et al., 2014). Possibly, this may be provoked by light-mediated resetting of the SCN circadian clock and inhibition of melatonin release (Boivin et al., 1996; Zeitzer et al., 2000). Additionally, in modern societies roughly 21% of employees work outside the regular working hours or in shifts, which has been associated with various adverse health outcomes (Karatsoreos et al., 2011; Parent-Thirion et al., 2016; Streng et al., 2022). Shift work is believed to affect the alignment of an individualâs behavioral cycle with both external and endogenous rhythms resulting in a loss of phase coherence between two or more circadian rhythms (Boudreau et al., 2013; Skene et al., 2018). Numerous epidemiological studies have demonstrated a higher prevalence of various disorders in shift workers â from impaired mental health to metabolic syndrome, obesity, cardiovascular disease, autoimmune disorders, and cancer (Chellappa et al., 2020; Davis et al., 2001; de Bacquer et al., 2009; di Lorenzo et al., 2003; Ellingsen et al., 2007; Karlsson et al., 2001; Kawachi et al., 1995; Li et al., 2022; Magrini et al., 2006; Schernhammer et al., 2003). Circadian misalignment can also be caused by travelling across several time zones leaving us with sleep problems, fatigue, cognitive impairments, and gastrointestinal issues summarized as jetlag disorder. During jetlag, the bodyâs internal time is misaligned to local geophysical time, and the circadian clock network needs some time to entrain to the new time zone (Diekman and Bose, 2018; Kiessling et al., 2010; McGuckin et al., 2014; Roach and Sargent, 2019; Wright et al., 1983).\n\n\nLevels of organization of the mammalian circadian clock system\n\nThe SCN constitutes a network of interconnected neural clocks with distinct periods in vitro (Honma et al., 2004; Welsh et al., 1995). However, a robust circadian output is observed probably arising from strong inter-cellular coupling within the SCN. Non-SCN tissue clocks show fast dampening of circadian oscillations in vitro likely due to weak inter-cellular coupling and the high dependence on systemic signals to keep synchrony (Yoo et al., 2004). Coupling can be observed on different levels such as systemic (inter-tissue) and intra-tissue which can be further subdivided into inter-cellular and molecular. All contribute to the generation or maintenance of coherent rhythms in behavior and physiology.\n\nThe SCN synchronizes the clock network via numerous signals. Vice versa, rhythmic signals from the periphery feedback to the SCN to stabilize and fine-tune the clock system. This type of crosstalk occurring between different tissue clocks is known as systemic coupling (Pilorz et al., 2020). The autonomic nervous system and the endocrine system are important routes utilized by SCN to deliver humoral and neural signals to peripheral tissues. Thereby, the SCN regulates changes in the blood supply, kidney filtration rates, hormone secretion, sensitivity for hormones, and insulin sensitivity in a time-dependent manner (Buijs et al., 1999, 1993; Cailotto et al., 2005; Cui et al., 2001; Dai et al., 1997; Deering and Coote, 2000; Kalsbeek et al., 1993; Kalsbeek and Strubbe, 1998; Vujovic et al., 2015). The best studied circadian hormones are glucocorticoids and melatonin, and both act as systemic synchronizing signals (Cheifetz, 1971; de Kloet and Sarabdjitsingh, 2008; Klemcke et al., 1989; Lincoln et al., 1982; Oster et al., 2006; Perlow et al., 1981; Redman et al., 1983). On the other hand, systemic signals derived from peripheral tissues reach the SCN for a proper synchronization of physiological rhythms (Balsalobre et al., 2000; Buijs et al., 1999; Gerber et al., 2013; Guo et al., 2005; Oster et al., 2006; Redman et al., 1983). For this reason, various hormone receptors are present in the SCN modulating and fine-tuning internal circadian timing. Intriguingly, the hepatokine fibroblast growth factor 21 (FGF21), a starvation signal secreted from the liver into the blood stream, can affect the SCN, thereby modulating several physiological functions (Bookout et al., 2013). Another example, leptin, an adipocyte derived peptide, conveys the metabolic state of peripheral tissues to the SCN. Interestingly, leptin receptor deficient rats show disrupted circadian rhythms of food intake (Li et al., 2012). The stomach-derived peptide hormone ghrelin can modulate food anticipatory activity through its action on the mediobasal hypothalamus (MBH) (Merkestein et al., 2014; Wang et al., 2018). Thus, systemic coupling mechanisms are crucial to efficiently synchronize circadian rhythms across the entire body.\n\nOn tissue level, one can discriminate between inter-cellular and molecular coupling. For inter-cellular coupling, single cells within a tissue communicate with each other using various signaling mechanisms, thereby, creating internal synchrony across the organ. Coupling between different cells of the same tissue is best described for the SCN. Here, gap junctions, paracrine signals, chemical synapses, and electrical signaling are used to keep SCN cell rhythms synchronized and aligned with each other (Maywood et al., 2011; Rash et al., 2007; Yamaguchi et al., 2003). Tetrodotoxin-mediated inhibition of neurotransmission results in reduced amplitudes of circadian clock gene expression (Yamaguchi et al., 2003). Strong inter-cellular coupling in the SCN results in robust rhythms which can also compensate genetic clock disruption for which peripheral tissues are much more sensitive (Liu et al., 2007). In line with this, SCN explants show circadian oscillations for weeks whereas circadian oscillations diminish in peripheral tissue explants after some days suggesting much weaker inter-cellular coupling (Yoo et al., 2004). However, hepatocyte clocks in SCN ablated mice show fast and stable entrainment to daytime feeding indicating that peripheral tissue clocks have to be coupled to a certain extent (Saini et al., 2013). It remains elusive whether the synchronized circadian oscillations in liver arise through the integration of systemic signals or by coupling on the tissue level. Hepatocyte cell culture experiments suggest that there is weak coupling between the cells since desynchronization over time is slower than in a simulation of a model where no coupling is assumed. Coupling in the periphery is weak and locally more restricted compared to what is shown for the SCN (Guenthner et al., 2014), and the underlying mechanisms are largely unknown.\n\nMolecular coupling within a rhythmic cell is established through interlocked transcriptional- translational feedback loops (TTFLs) regulating rhythmic transcription and post-translational modifications controlling the stability of clock proteins (Buhr and Takahashi, 2013). Within each rhythmic cell, the molecular clock has a normal phase distribution. The new transcriptional cycle of the molecular clock starts once brain and muscle ARNT like protein 1 (BMAL1): circadian locomotor output cycles kaput (CLOCK) heterodimers bind to E-box promotor elements in period1â3 (Per1â3) and cryptochrome1/2 (Cry1/2) genes inducing their transcription in the morning (Kume et al., 1999; Shearman et al., 1997). Later, towards the night, PER and CRY heterodimers are translocated to the nucleus where they repress their own transcription by inhibiting binding of BMAL1:CLOCK to E-boxes (King et al., 1997; Konopka and Benzer, 1971; Reddy et al., 1984; van der Horst et al., 1999; Zheng et al., 1999). Beside the E-box elements, other response elements such as D-boxes and retinoic acid receptor-related orphan receptors elements (ROREs) are modulating and stabilizing circadian oscillations (Akashi and Takumi, 2005; Mitsui et al., 2001; Nakajima et al., 2004; Ohno et al., 2007; Preitner et al., 2002; Sato et al., 2004; Ueda et al., 2002; Yamaguchi et al., 2000). D-boxes are bound among others by the transcriptional repressor nuclear factor interleukin-3-regulated protein (NFIL3, also known as E4BP4) and the transcriptional activator D-box binding protein (DBP) modulating the expression of, e.g., Per1â3, repressive reverse-erythroblastosis virus α/β (Rev-Erbα/β), and activating retinoic acid receptor-related orphan receptors (Rorα/β/γ) (Mitsui et al., 2001; Ohno et al., 2007; Ripperger and Schibler, 2006; Yamaguchi et al., 2000). Repressing REV-ERB α/β and activating RORα/β/γ proteins compete for binding to ROREs modulating the expression of Bmal1, Clock and Cry1 (Guillaumond et al., 2005; Nakajima et al., 2004; Preitner et al., 2002; Sato et al., 2004; Ueda et al., 2002). The interlocked feedback-loops result in ~24- hour oscillation of gene expression and repression. Importantly, such oscillations are further stabilized and fine-tuned by chromatin remodeling, post-transcriptional, and post-translational modifications impacting the stability of distinct clock mRNAs/proteins (Doi et al., 2006; Duong and Weitz, 2014; Gallego and Virshup, 2007; Grimaldi et al., 2007; Nakahata et al., 2008; Grimaldi et al., 2009; Kojima et al., 2011; PreuÃner et al., 2014). Together, these lead to a stable phase relationship/coherence between the different genes. In general, expression of Bmal1 is almost anti-phasic to the expression of Per2, Nr1d2 and Cry2 (10â12 h), whereas expression of Nr1d1 is only phase-delayed by 6â10 h. Interestingly, the peak expression of Bmal1 and Cry1 differs only between 2â4 h (Mure et al., 2018). Of note, the phase relationship of the clock genes to each other is tissue- and species-specific (Harbour et al., 2014; KorenÄiÄ et al., 2014; Mure et al., 2018; Pett et al., 2018; Yeung et al., 2018). Moreover, there is a stable phase relationship of single-tissue clocks to each other. In general, non-SCN tissues are phase-delayed compared to the SCN. Adrenal gland clocks show a small phase delay compared to those in the liver, whereas liver, adipose tissue, and muscle are similarly phased (KorenÄiÄ et al., 2014; Mure et al., 2018; Yang et al., 2006). Since all tissue clocks contribute to generate coherent circadian rhythms, the phase coherence within the cell, between the cells and among the tissues is potentially important to maintain homeostasis.\n\nGenetic mutations or knockouts (KO) of circadian clock genes lead to the development of various diseases ranging from sleep disorders to cardiovascular, mental, and metabolic deteriorations (Dibner and Schibler, 2015; Kiehn et al., 2017; Valenzuela et al., 2016). In recent years, evidence has been accumulated on the physiological relevance of distinct tissue clocks. Liver-specific Bmal1 KO results in abolished rhythms in glucose regulatory genes creating problems in the maintenance of blood glucose levels especially during fasting periods (Lamia et al., 2008). Elevated levels of blood lipids were observed in mice with hepatocyte-specific ablation of REV-ERBα/β (Bugge et al., 2012; Cho et al., 2012). Skeletal muscle clock regulates glucose metabolism. Mice lacking a functional muscle clock show defective insulin-stimulated glucose uptake attributed to impaired glucose transporter 4 (GLUT4) translocation to plasma membrane (Dyar et al., 2014; Harfmann et al., 2016). In adipose tissue, circadian clocks regulate several metabolic processes in a time-of-day dependent manner. Adipocyte-specific Bmal1 KO results in obesity and increased rest-phase food intake probably related to altered fatty acid signaling to the hypothalamus (Paschos et al., 2012). In heart, genetic ablation of Bmal1 results in perturbed systolic function and abnormal glucose utilization (Young et al., 2014). Lastly, adrenal cortex-specific Bmal1 KO mice display dampened glucocorticoid and locomotor activity rhythms (Dumbell et al., 2016; Son et al., 2008). Of note, local tissue clocks are not sufficient to drive the local transcriptome independently (Koronowski et al., 2019). Altogether, these studies emphasize the importance of specific tissue clocks, but they also show that coherence among different tissue rhythms is important to maintain whole body homeostasis.\n\n\nFrom a circadian-resonant to a chronodisrupted health state\n\nCircadian clocks throughout our body integrate external and internal rhythmic signals to drive coordinated rhythmic physiological and behavioral outputs. When these inputs are appropriately aligned, the body is in a resonant or synchronous state which enhances resilience to pathogenic insults and thus, stabilize health and wellbeing. However, when zeitgeber input is perturbed, e.g., the occurrence of temporal stimuli is temporally shifted or the duration is altered, chronodisruption may emerge. With regard to different zeitgebers, the LD cycle is considered as the most potent zeitgeber for entraining circadian and seasonal rhythms in most species. In real life, a misalignment of geophysical time and circadian rhythms is observed after rapidly crossing different time zones. This provokes jetlag disorder with disrupted sleep/wake cycles, fatigue and cognitive impairments until the internal clock system is entrained to the new geophysical time. While for the general population jetlag occurs only once in a while, which has little persisting health effects, long-distance flight personnel experiences repeated jetlag which may cause long-term impairments. Most people in modern societies, on the other hand, are exposed to artificial light during natural dark periods. Increasingly more people are experiencing LAN during work in shifts (reviewed in detailed by Touitou et al., 2017). Shift workers commonly have inefficient and poor quality sleep causing fatigue (Paech et al., 2010). Moreover, forced activity and sleep deprivation in the normal rest phase strongly impacts the immune system (Irwin et al., 2006), and shift workers are more likely to develop chronic medical conditions like cardiovascular diseases (Ha and Park, 2005), metabolic syndrome (Cheng et al., 2021), psychological disorders (reviewed by P. Cheng & Drake, 2018) and others. Simulations of shift work and abnormal LD cycles in laboratory settings are used to investigate the mechanisms of chronodisruption and will be described in detail in the next sections. Usually feeding and social behavior are also almost exclusively scheduled during active phases of the circadian cycle. They serve as potent entraining stimuli to some CNS and peripheral tissue clocks. Availability of food round-the clock and energy requirement during shiftwork leads to mistimed food intake in humans that manifests into various chronic metabolic diseases including obesity and type-2 diabetes. Tissues clocks that are strongly influenced by feeding signals, e.g. liver or kidney, can be uncoupled from the SCN inducing inter-tissue desynchrony (Damiola et al., 2000; Hara et al., 2001; Stokkan et al., 2001). All of the above described real-life situations result in chronodisruption which promotes metabolic impairments but also increases the prevalence for major depression, cardiovascular disease, autoimmune disorders, and certain cancers (Davis et al., 2001; de Bacquer et al., 2009; di Lorenzo et al., 2003; Ellingsen et al., 2007; Erren and Reiter, 2009, 2013; Karlsson et al., 2001; Kawachi et al., 1995; Li et al., 2022; Magrini et al., 2006; Schernhammer et al., 2003). Although the concept of chronodisruption is established, its pathogenic mechanism remains largely elusive. Chronodisruption can occur at different levels, i.e., (1) a mismatch of external time and internal circadian rhythms (external-internal desynchrony), (2) phase incoherence between different tissue clocks (inter-tissue desynchrony), (3) phase incoherence between cells of the same tissue (cellular desynchrony) and, (4) phase incoherence at the molecular level (molecular desynchrony) (Figure 2). In the following sections we will describe these different levels of desynchrony and outline what is known about their physiological consequences.\n\nMisaligned zeitgebers like artificial lighting conditions, mistimed food intake, or activity in the rest phase create dissonance between the internal circadian timing system and geophysical time â a state somewhat vaguely termed as chronodisruption. Desynchrony of circadian rhythms can occur at various levels â from misalignment of internal and external time (as during jetlag) to alterations in the coordination of different clock gene expression rhythms at the cellular level. It is still poorly understood how the different levels of circadian desynchrony contribute to the adverse health effects of chronodisruption. Figure created with BioRender.com.\n\n\nDesynchrony between external and internal rhythms\n\nMisalignment between zeitgebers and internal circadian rhythms evokes chronodisruption (Figure 2). This state is often caused or enhanced by perturbed zeitgebers such as phase-shifted LD cycles, activity in the inactive phase (shift work), or mistimed food intake, all of which have been suggested to promote metabolic and psychological ailments.\n\nMismatch between geophysical time and internal biological timing, as observed in jetlag conditions, causes physiological behaviors like sleep, hunger and defecation to occur according to internal timing at non-regular times of the day. However, people in aviation industries who are exposed to repeated jetlag report chronic sleep-inefficiency associated physiological (reviewed in Arendt and Marks, 1982) and cognitive health problems (Cho et al., 2000). Simulated chronic jetlag conditions induce obesity in mice (Oike et al., 2015). However, body weight gain is observed only in advanced but not in delayed LD cycles (Casiraghi et al., 2016). Chronic jetlag retains oscillations, but induces phase shifts in clock gene expression rhythms in SCN and liver of mice (Iwamoto et al., 2014). In laboratory settings, rodents are exposed to non-24-hour LD cycles (T-cycles) to investigate the mechanisms of entrainment. Chronic exposure to short T-cycles (4 h light: 4 h darkness (4:4 LD)) â but not long T-cycles (18:18 LD) â decreases life span. Interestingly, clock deficient mice show no differences in mortality rates under long and short T-cycles. The authors suggest that the combination of specific LD cycles and perturbations of the circadian clock network results in impaired homeostatic regulation impacting longevity (Park et al., 2012). In mice, T-cycles, both 11.25:11.25 LD and 13.5:13.5 LD, reduce energy efficiency evidenced by higher food intake despite no body weight gain. Animals increase their food consumption during the light phase. Thereby, mice predominantly utilize carbohydrates to fuel the body which hints to an impaired metabolic homeostasis (West et al., 2017). Under 11:11 LD cycles, rats show dyslipidemia and lowered expression levels of proteins critical for insulin signaling as well as increased levels of enzymes involved in gluconeogenesis in liver indicating metabolic disruptions induced by forced desynchrony (de Oliveira et al., 2019). Non-24-hour LD cycles result in abolished rhythmicity in heart rate and blood pressure (Molcan and Zeman, 2017; West et al., 2017). In humans, circadian misalignment evokes an overall increase in blood pressure (Morris et al., 2016; Scheer et al., 2009). Of note, the loss of rhythmic LD cycles as experienced during LAN or constant light (LL) conditions also impacts circadian outputs such as food intake patterns, energy expenditure and corticosterone rhythms promoting elevated body weight, metabolic impairments and depressive-like behavior (Coomans et al., 2013; Fonken et al., 2012, 2010; Tapia-Osorio et al., 2013).\n\nShift workers are forced to be active during their normal rest phase. Such work regimes are often accompanied by food consumption at inappropriate times. Thus, shift workers are simultaneously exposed to various chronodisrupters such as LAN, mistimed food intake and mistimed behavioral rhythms. Such employees show dampened cortisol and testosterone rhythms (Touitou et al., 1990). Furthermore, shift workers exhibit higher triglyceride and lower HDL (high density lipoprotein) cholesterol levels and higher abdominal obesity than regular day-time workers (Karlsson et al., 2003). Night shift work can be simulated in rodents by forced activity in the normal resting phase. This shifts activity and food intake into the resting phase. Consequently, daily blood glucose rhythms are abolished, triglyceride rhythms are reversed and corticosterone levels are transiently increased (Salgado-Delgado et al., 2008). In laboratory studies, shift work can be simulated by imposed 28-hour days in humans. In such conditions, sleep and food intake patterns are 12 h out of phase from the habitual times within a few days. On these (misaligned) days, participants exhibit completely reversed cortisol rhythms and increased arterial pressure. In addition, they show impaired metabolic homeostasis with increased blood glucose despite increased insulin levels compared to the circadian aligned baseline day (Scheer et al., 2009). Interestingly, daytime-restricted food consumption prevents the dissociation of body temperature rhythms, a measure of the central clock, and peripheral glucose rhythms. In turn, this prevents detrimental outcomes in glucose tolerance especially in the biological morning in the 28-hour shift work protocol (Chellappa et al., 2021). The studies show that already short-term chronodisruption generates adverse metabolic and cardiovascular effects. In addition to metabolic and psychological repercussions, far more detrimental effects of chronodisruption induced by shiftwork are carcinogenesis and malignancy. Numerous studies have shown cancer as a consequence of such a work regime (Davis et al., 2001; Mazzoccoli et al., 2011; Schernhammer et al., 2003; Viswanathan et al., 2007). However, a recent meta-analysis shows overall no significant association between the two (Dun et al., 2020). The meta-analysis considers factors like geophysical region, time and gender differences to compare data between different cohorts; however, factors like type of shift work, food intake habits and general health condition of the individual may also play a role in the cancerogenic effects of shift work. Not only shift workers, but even larger parts of the working force face discrepancy in their sleep/wake cycles between workdays and free days imposing a state termed social jetlag. This regular shift in sleep/wake phasing likely represents a condition of chronic circadian misalignment. People with strong social jetlag show higher cortisol levels and increased resting heart rates (Rutters et al., 2014). Social jetlag is also associated with increased body mass index and metabolic syndrome (Roenneberg et al., 2012; Parsons et al., 2015). Unhealthy obese subjects show elevated levels of the inflammatory marker C-reactive protein and the obesity-related biomarker glycated hemoglobin with increased social jetlag (Parsons et al., 2015). These data show that chronodisruption impairs metabolic homeostasis and contributes to aggravation of the medical conditions.\n\nFood intake, being an important zeitgeber especially for peripheral tissue clocks, disturbs internal resonance of rhythms when occurring at the wrong time-of-day. On the long-term, this may result in obesity and other metabolic disorders. Daytime restricted feeding in nocturnal mice uncouples the liver clock from the SCN (Damiola et al., 2000; Hara et al., 2001; Stokkan et al., 2001). Food intake only during the light phase also phase-shifts the acrophase of genes involved in lipid homeostasis and bile acid metabolism which may cause adverse metabolic outcomes (Cui et al., 2022). High-caloric food is shown to disrupt daily food intake patterns and rhythms in clock as well as metabolism related genes. Such disruption can be prevented when the high-caloric diet is only available during the night in nocturnal mice. This intervention also reduces metabolic diseases like hepatic steatosis and hypercholesterolemia observed in ad libitum fed mice (Chaix et al., 2014).\n\nIn modern societies, several zeitgebers are misaligned to each other and to the endogenous circadian clock. When mice are subjected to a combined 14:14 LD with a 12:12 fasting/feeding (FF) protocol they become dynamically exposed to aligned and misaligned zeitgeber conditions. Surprisingly, mice transiently show weight gain and impaired glucose tolerance on the day of aligned zeitgeber input (i.e., when feeding coincides with the dark phase) compared to the day of misaligned zeitgeber input (feeding during the light phase). Such zeitgeber misalignment further evokes a change in the phasing of different tissue clocks (Heyde and Oster, 2019). In conclusion, misalignment between internal and external rhythms is associated with alterations in metabolic and psychological states and may promote obesity, diabetes, cardiovascular diseases, depression, anxiety, and cancer.\n\n\nInter-tissue desynchrony\n\nDesynchrony caused due to external-internal misalignment can impinge further than just the systemic level by inducing phase incoherence between different tissue clocks and circadian tissue outputs. Tissues differ in their susceptibility to external and hormonal signals from each other. Thus, they adapt to changed input rhythms at a different pace inducing misalignment between tissue clocks and rhythms â a state termed as inter-tissue desynchrony.\n\nCircadian homeostasis is maintained by communication and synchronization between and among central and peripheral clocks. Ablation of the SCN clock eliminates diurnal variation in clock gene expression in peripheral tissues including liver, kidney, heart, skeletal muscle, and spleen. Parabiosis of SCN-lesioned and intact animals re-induces day-night variation in SCN-lesioned mice in liver and kidney but not in heart, skeletal muscle and spleen. This emphasizes the differential dependence on blood-borne signals of distinct tissue clocks (Guo et al., 2005). However, recent studies observed that in LD the SCN clock is dispensable for clock network synchronization but becomes necessary under constant darkness (DD) conditions. Animals with an ablated SCN clock show internal desynchrony, i.e., phase incoherence between different tissue clocks, within a few days under DD conditions (Husse et al., 2011; Izumo et al., 2014; Kolbe et al., 2019; van der Vinne et al., 2018). However, effects of internal desynchrony on body weight seem to be dependent on the Cre driver line used (Kolbe et al., 2019; van der Vinne et al., 2018). Of note, internal desynchrony and weight gain are prevented by maintaining the animals on time-restricted feeding schedules under DD conditions highlighting the importance of food intake timing for peripheral clock synchronization (Kolbe et al., 2019).\n\nMany studies use constant light (LL) or repeated jetlag conditions to investigate the effects of abnormal light cycles on different tissue clocks. LL conditions not only weaken the central clock but also disrupt phase alignment between different tissues. Mice subjected to LL show immediate dampening in the amplitude of SCN rhythms (Coomans et al., 2013). LL housing maintains rhythmic PER2 oscillations in liver and kidney but distinctly reduces amplitude and broadly distributes phases of its rhythm. In submandibular gland, this rhythm is lost in a significantly higher number of animals (Hamaguchi et al., 2015). On transcriptional level, the liver and colon display abolished diurnal expression of clock genes (Polidarová et al., 2011). In addition, to clock outputs, LL eliminates rhythmicity of numerous genes involved in lipid metabolism in liver and white adipose tissue (WAT). Decreases in amplitude and average levels of gene expression are more pronounced in the liver compared to WAT, indicating higher sensitivity of metabolic and clock gene transcription in the liver to constant light conditions (Yamamuro et al., 2020). Upon jetlag, distinct tissue clock genes display differences in entrainment speed to the new LD schedule. Here, the misalignment caused is temporary and is resolved when tissue clocks re-align with the new LD cycle. SCN and adrenal rapidly adapt to the changed lighting schedule whereas liver and kidney re-entrain at a slower pace, with the pancreas clock being the slowest (Kiessling et al., 2010). PER1 oscillations in the arcuate nucleus quickly entrain upon a phase-delaying jetlag whereas the paraventricular nucleus (PVN) and pineal gland are faster to re-entrain upon a phase-advancing jetlag in in vitro studies (Abe et al., 2002). These studies indicate that it is likely that jetlag induces internal desynchronization not only between central and peripheral clocks but also between clocks within the central nervous systems. The sleep/wake cycle is one of the behavioral outputs driven by the SCN. Two weeks of timed-sleep restriction, mimicking human night shiftwork, have only moderate effects on the levels of clock gene expression in the SCN but markedly change expression levels and phasing in the liver. Moreover, circadian liver transcriptome data show pronounced changes especially in glucose metabolism related genes which results in impaired performance in gluconeogenesis and increased glycogen storage in the beginning of the dark phase. Time-restricted feeding to the normal active phase is able to prevent the effects of time-restricted sleeping on hepatic clock gene expression and metabolic outcomes (Barclay et al., 2012).\n\nPeripheral clocks show tissue-specific pace in feeding-induced phase resetting. Upon daytime restricted feeding, phases of the clocks in liver, kidney, heart, lungs and pancreas in mice are transiently misaligned to each other. However, all these tissues entrain to the new feeding schedule within a week (Damiola et al., 2000; Hara et al., 2001; Opperhuizen et al., 2016; Stokkan et al., 2001). The dorsomedial hypothalamus also entrains to daytime restricted feeding (Verwey and Amir, 2011). The SCN clock, however, stays aligned to the LD cycle (Damiola et al., 2000; Hara et al., 2001; Opperhuizen et al., 2016; Stokkan et al., 2001). In contrast, clock gene rhythms are abolished in lateral hypothalamus, skeletal muscle and arcuate nucleus in rats fed during the light period (Opperhuizen et al., 2016; Wang et al., 2017). The studies show that feeding time has differential effects on SCN and non-SCN CNS as well as peripheral clocks, thus, weakening inter-tissue phase coherence â which likely interferes with the maintenance of metabolic homeostasis.\n\nIn addition to the mistiming of food intake, changes in diet composition can induce internal desynchrony. High-fat diet (HFD) feeding is known to disrupt behavioral and physiological rhythms, but the mechanisms are still not fully understood. It is likely that internal desynchronization of circadian rhythms plays an important role. Long-term HFD feeding alters clock gene expression rhythms in liver and adipose tissue (Kohsaka et al., 2007; Yamamuro et al., 2020). Minimal effects on clock gene rhythms were observed in MBH, a center which controls hunger and satiety, and the medial prefrontal cortex, a region involved in cognitive functions (Kohsaka et al., 2007; Tognini et al., 2020). Of note, HFD reduces the total number of oscillating genes, significantly advances the acrophase and dampens the amplitude of rhythmic genes, but not clock genes, in the SCN demonstrating that the SCN is susceptible to food signals (Tognini et al., 2020). The liver clock is not only susceptible to feeding time but also to food composition. In vitro studies confirm a high susceptibility of the liver clock to food-related signals whereas other tissue clocks such as lung, pituitary and arcuate complex are less receptive (Pendergast et al., 2013). Food related signals which modulate clock resetting include insulin (Chaves et al., 2014; Crosby et al., 2019; Sato et al., 2014; Sun et al., 2015; Tahara et al., 2011), oxyntomodulin (Jorgensen et al., 2007; Landgraf et al., 2015), and many others (reviewed in Reinke and Asher, 2019). Additionally, glucose, amino acids and other metabolites can also phase-shift or entrain circadian clocks (reviewed in Froy, 2007). As such, it can be speculated that re-entrainment of a distinct tissue clock is dependent on its susceptibility to clock modulators. Altogether, the timing of food intake and food composition can rapidly lead to inter-tissue phase incoherences which might be transient or permanent.\n\nUnder diverging zeitgeber input (14:14 LD cycle combined with a 12:12 FF cycle), liver and epididymal WAT clocks align with the FF schedule whereas adrenal and SCN tissue clocks are highly impacted by the extended LD cycle. Thus, animals suffer from internal desynchrony between distinct tissue clocks and circadian output. Interestingly, mice do not gain weight but show circulating body weight changes dependent on the transient state of zeitgeber (mis)alignment (Heyde and Oster, 2019). Such internal desynchrony is already observed after four days of 14:14 LD/12:12 FF conditions and is less pronounced when feeding is restricted to the dark phase under 14:14 LD conditions (Heyde and Oster, 2022).\n\nTaken together, changes in the external environment or (forced) changes in the behavioral cycle result in phase incoherences between different oscillators which may cause disruption of rhythmic output eventually resulting in metabolic impairments. However, more studies investigating the effect of phase incoherences between certain tissues are needed to fully elucidate the physiological consequences.\n\n\nIntra-tissue desynchrony\n\nTissues contain numerous cell types with each cell containing an autonomous molecular clock. Synchronized cells give rise to overt circadian rhythms on the tissue level. Cells integrate several signals to produce coherent rhythms. Phase incoherences of single cellular clocks within a tissue cause intra-tissue desynchrony. Moreover, at the single-cell level the phasing of distinct clock gene rhythms to each other can be disrupted giving rise to molecular desynchrony (Figure 2). Current standard techniques face limitations in distinguishing between cellular and molecular levels of chronodisruption. Thus, in this section we describe both levels together as intra-tissue desynchrony.\n\nWith regards to desynchrony caused between different cells the best-studied tissue is the SCN. The SCN consists of two major regions with light responsive neurons in the ventrolateral and light unresponsive ones in the dorsomedial part (Van den Pol, 1980). Abrupt delaying or advancing of LD rhythms rapidly causes incoherence of synchronous rhythms of clock genes between the two SCN regions. Clock genes in the ventrolateral SCN are quick to show large shifts compared to the dorsomedial region which shifts slower and takes a longer time to resynchronize to the new lighting schedule (Nagano et al., 2003). Shifts in lighting conditions also disrupt phase relation, i.e., peak phase differences between dorsal and ventral parts of the SCN. After a phase-delaying shift in the LD cycle, peak phase differences between the regions increase but return to pre-phase shift conditions within three days. In contrast, a phase-advancing shift in the LD cycle leads to reversed phase relations between the regions and the pre-shift state is reached only after six days (Nakamura et al., 2005). A recent study with imposed delays in the LD cycle investigated Per2 expression at a single-cell level throughout the SCN. The ventral part contained about 40% fast phase-shifting and 60% slow phase-shifting neurons while the dorsal SCN almost exclusively comprised slow-shifting neurons (van Beurden et al., 2022).\n\nAs mentioned above, phase incoherence can also occur at the level of the molecular clock work itself. In mice, in the SCN Per1 and Per2 rhythms more rapidly react to LD shift advances compared to Cry1. Whereas all three genes react rapidly to LD delays (Reddy et al., 2002). Similar transient dissociation is observed between Per1 and Bmal1 rhythms in mice exposed to a single light pulse during DD conditions. Per1 is instantaneously phase-delayed together with activity onset while Bmal1 shifts more gradually in parallel with activity offset (Ono et al., 2017).\n\nDesynchrony within clock gene expression is also observed in peripheral tissue clocks. HFD attenuates amplitude of clock gene profiles in MBH, adipose and liver tissue. In the MBH, although Per2 and Bmal1 show robust rhythms, Clock RNA expression is completely abolished. In contrast, Clock is diurnally rhythmic in both fat and liver but with altered amplitude. Additionally, Bmal1 is attenuated throughout the 24-hour day, but Per2 shows decreased amplitudes only in the dark phase (Kohsaka et al., 2007). Notably, not only incoherence in phasing of clock genes but also differences in amplitude of their rhythm may contribute to desynchrony. However, it has to be investigated whether changes in amplitude result from impaired clock gene expression on a single-cell level or broader averaging over phase distributed cells. Differential pace of entrainment is observed for distinct clock genes upon jetlag conditions. In the SCN, somatosensory cortex and adrenals, Per1 and Per2 rapidly entrain to the new LD schedule whereas entrainment of Dbp and Nr1d1 is slower. Interestingly, in the liver, Per1 and Dbp re-entrainment is slower compared to Per2 expression rhythms. Bmal1 shows the slowest pace of entrainment in all tissues. Of note, in the pancreas, Nr1d1 is quickly re-adjusted while Per1 and Per2 are slower (Kiessling et al., 2010). Under zeitgeber intervention conditions, a higher degree of phase incoherence between clock gene expression peaks is observed under LD-28/FF-28 compared to LD-28/FF-24 conditions in liver, adrenal, and epididymal WAT (Heyde and Oster, 2022).\n\nExploring circadian characteristics on the single-cell level within a tissue is technically challenging and not thoroughly studied yet. In contrast, more evidence on phase incoherence between different clock genes within a tissue is arising. Both levels of intra-tissue desynchrony might impinge on the circadian output of the tissue affecting health, but more studies are needed to elucidate the specific impact.\n\n\nTherapeutic approaches and future directions\n\nCircadian clock network coordination is disrupted by alterations in zeitgeber input rhythms. Numerous studies show that such perturbation may result in metabolic, mental, and cardiovascular impairments. It is important to consider that these ailments arise from circadian misalignment which occurs at different levels of organization. However, causality has to be proven. Disruption of circadian rhythms usually emerge due to misalignment between external time cues and internal time. Such misalignment promotes internal desynchrony between distinct tissue clocks. Although inter-tissue desynchrony is an established concept, experimental evidence is still sparse and, therefore, the physiological outcomes are not well understood. It can be speculated that desynchrony between cellular clocks of the same tissue contributes to dampening of circadian tissue output, which consequently impacts homeostasis. Again, so far this has not been proven directly. Finally, it remains elusive whether such perturbed circadian output is caused by impaired rhythmic transcription and translation at the cellular level or by phase incoherence between cells within the tissue.\n\nIt will be important to understand the mechanisms and effects of internal desynchrony at different levels to develop targeted therapeutic approaches that can prevent chronodisruption-associated diseases. Differentiating the physiological impact of desynchrony on each level may be technically challenging and good experimental paradigms dissecting the different forms of desynchrony are still sparse. As one example, the development of transgenic mice harboring luciferase reporters coupled to a clock gene promotor enabled researchers to investigate effects of zeitgeber intervention in real-time in freely moving animals (Ono et al., 2015; Saini et al., 2013; Yamaguchi et al., 2016). Recently, fluorescent clock-reporters were used to track circadian oscillation in SCN neuronal sub-populations in vivo (Mei et al., 2018). However, so far it is not possible to study changes on single-cell levels in vivo. In vitro, this is overcome by introducing two reporters. Here, one fluorescent clock-reporter gives information on the phase of the clock while the second fluorescent reporter is used to track single cells (Manella et al., 2021). Future developments in this direction might enable us to study the impact of zeitgeber input on single cells in vivo and may help in understanding the physiological effects.\n\nTo date, therapeutical approaches to increase internal synchrony are limited and not targeted but instead impact clocks throughout the body which may not in all cases be beneficial. Future studies should investigate whether modulation or weakening of distinct tissue clocks may be beneficial under chronodisruptive zeitgeber conditions. First findings suggest that weakening of the SCN clock or intra-tissue coupling might be beneficial for re-entrainment to shifted LD cycles; however, whole-body homeostasis was not studied (An et al., 2013; Yamaguchi et al., 2013; Pilorz et al., 2014). Bright-light therapy, affecting the SCN clock, reduces the misalignment of geophysical time and internal rhythms. It is shown to improve circadian rhythms and poor sleep quality in elderly, Alzheimer patients and people with major depression (Yamadera et al., 2000; Terman et al., 2001; Neikrug et al., 2012; Lam et al., 2016; Rubiño et al., 2020). Humoral signals impact on specific tissue clocks. However, we know too little about how the different tissue clocks are synchronized. Melatonin increases sleep quality and can entrain blind people to a 24-hour day (Hack et al., 2003; Redman, 1997; Sack et al., 2000). Melatonin-mediated effects were attributed to the inhibiting and phase-shifting effect on SCN activity, but recently its effects on peripheral clock gene expression were shown (Jung-Hynes et al., 2010; Torres-Farfan et al., 2006). Timed administration of melatonin might be beneficial to treat shift work-associated sleep disorders, but it is unknown how the circadian clock system is affected (Carriedo-Diez et al., 2022). One of the best studied hormones affecting tissue clocks are glucocorticoids. Glucocorticoid receptors are expressed in almost all tissues. While most tissue clocks can be reset by glucocorticoids, the SCN clock is unresponsive due to low receptor expression (Rosenfeld et al., 1988; Reddy et al., 2007; Kiessling et al., 2010; Balsalobre et al., 2000). Glucocorticoids also regulate the expression of genes involved in glucose and lipid regulatory pathways in liver and adipose tissue, thereby controlling the maintenance of energy homeostasis (Guia et al., 2014). Time-restricted feeding has been shown to prevent some metabolic impairments under chronodisruptive conditions, but not all tissues are susceptible to food-related signals. Energy state-related hormones such as insulin, leptin, glucagon, ghrelin, and adiponectin are capable to impact clocks but mainly control energy homeostasis (Martinez-Merlos et al., 2004; Prosser and Bergeron, 2003; Sun et al., 2015; Tahara et al., 2011; Tsang et al., 2020; Yannielli et al., 2007). Rhythmic administration of adiponectin rescues diurnal variation in gene expression in the MBH and leads to body weight reduction (Tsang et al., 2020). These findings indicate that hormones may be useful to synchronize clocks and clock output in a tissue-specific manner which in turn ameliorates health outcomes. However, caution must be taken since most hormonal receptors are expressed in numerous tissues and, thus, it is impossible to target specific tissue clocks which in turn, may lead to undesirable side effects. Moreover, recent findings demonstrate that non-circadian signals alter tissue transcriptome rhythms without affecting the local clock. For example, elevated thyroid hormone levels result in dampened or abolished rhythms in glucose and lipid metabolism related genes in the liver but have no effect on clock gene expression (de Assis et al., 2022).\n\nFurthermore, despite exploring the potential use of hormones to improve internal synchrony, the efficacy of synthetic clock modulators gained attention in recent years. One of such, a REV-ERB agonist, may be useful to target metabolic impairments but it will also affect clocks throughout the body and side effects are not yet examined (Solt et al., 2012). The ROR agonist nobiletin enhances circadian rhythm amplitude and improves energy homeostasis preventing metabolic syndrome in diet-challenged mice (He et al., 2016). There are many more small-molecule modulators of the circadian clock and the therapeutic efficacy of such is proven or under investigation (reviewed in Ribeiro et al., 2021). To date, the clock machinery is assumed to be the same in all tissues, but there is evidence indicating that it is regulated in a tissue-specific manner (Mure et al., 2018). Global administration of small-molecule modulators of the circadian clock may have a differential effect on intra-tissue synchrony in which it will be beneficial for one tissue clock while worsening the phase relationship for another.",
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PubMed Abstract | Publisher Full Text\n\nYeung J, Mermet J, Jouffe C, et al.: Transcription factor activity rhythms and tissue-specific chromatin interactions explain circadian gene expression across organs. Genome Res. 2018; 28: 182â191. PubMed Abstract | Publisher Full Text\n\nYoo S-H, Yamazaki S, Lowrey PL, et al.: PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 5339â5346. PubMed Abstract | Publisher Full Text\n\nYoung ME, Brewer RA, Peliciari-Garcia RA, et al.: Cardiomyocyte-specific BMAL1 plays critical roles in metabolism, signaling, and maintenance of contractile function of the heart. J. Biol. Rhythm. 2014; 29: 257â276. PubMed Abstract | Publisher Full Text\n\nZeitzer JM, Dijk DJ, Kronauer R, et al.: Sensitivity of the human circadian pacemaker to nocturnal light: melatonin phase resetting and suppression. J. Physiol. 2000; 526(Pt 3): 695â702. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZheng B, Larkin DW, Albrecht U, et al.: The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature. 1999; 400: 169â173. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "155847",
"date": "19 Dec 2022",
"name": "Frank A.J.L. Scheer",
"expertise": [
"Reviewer Expertise Chronobiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEditorial Note from F1000Research â 25th April 2023: This peer review report was updated at the reviewer's request to add a Conflict of Interest statement in order to ensure full transparency.\nThis is a timely, comprehensive, and well-written review on circadian misalignment at different organismal levels and health consequences, with a primary review of animal experimental work supplemented with human work. The claims are well supported by references to original work. My comments are mostly minor.\nWhile this is done in many cases, it is important to specify the model and tissue. This is especially important because statements about timing of peaks and troughs may be opposite between nocturnal vs. diurnal mammals, and between SCN vs. extra-SCN oscillators. For example, statements such as \"circadian locomotor output cycles kaput (CLOCK) heterodimers bind to E-box promotor elements in period1â3 (Per1â3) and cryptochrome1/2 (Cry1/2) genes inducing their transcription in the morning\" need to indicate the model and tissue. Please do this throughout the manuscript.\n\nFigure 2, consider moving \"Inter-tissue desynchrony\" label to be with the third panel that is showing inter-tissue misalignment. Do you need the label âIntra-tissue desynchronyâ? This seems to refer to the same concept as âCellular desynchronyâ, i.e., misalignment between cells in the same tissue. Consider using the same terminology throughout the manuscript, and the term \"inter-cell\" to clarify that it is distinct from the intra-cellular/molecular misalignment.\n\nFor future directions, it will be helpful for the readers to lay out what type of studies and approaches will be most valuable to clarify the knowledge gaps. This is also true for the statement on page 9, âHowever, more studies investigating the effect of phase incoherencesâŠare neededâŠâ.\n\nIntroduction, first paragraph, replace \"period length\" with \"period\" and consider adding \"(cycle length)\" after \"period\".\n\nIntroduction, second paragraph, do the authors mean \"prevalence\" instead of \"incidence\"?\n\nIntroduction, second paragraph, âPossibly, this may be provoked by light-mediated resetting of the SCNâŠâ, clarify the rationale why SCN resetting, or melatonin suppression, would increase the risk for obesity or metabolic syndrome.\n\nIntroduction, second paragraph, âShift work is believed to affectâŠâ, the phase coherence doesn't need to be between two or more circadian rhythms but could also be between a circadian and a diurnal rhythm such as between the melatonin rhythm and the sleep/wake cycle or light/dark cycle.\n\nPage 4, âHowever, hepatocyte clocks in SCN ablated miceâŠâ, why would this show that peripheral tissue clocks are coupled? An alternative explanation could be that the feeding rhythm itself synchronizes hepatocytes, such that they are aligned even if they are not coupled among each other. Even though this alternative is recognized in the subsequent sentence, it will be helpful to acknowledge this in the first statement.\n\nPage 4, âWithin each rhythmic cell, the molecular clock has a normal phase distributionâ, consider rephrasing that ââŠthe components of the molecular clock have a phase distribution.â\n\nPage 6, âAll of the above described real-life situations result in chronodisruption which promotes metabolic impairments but also increases the prevalence for major depression, cardiovascular disease, autoimmune disorders, and certain cancersâ, most of these cited studies are observational studies where other factors may also be involved, besides chronodisruption, that cause the increased disease risk, such as differences in socioeconomic status and lifestyle which will be important to acknowledge and that therefore experimental studies are needed to directly test cause-and-effect relationships with risk factors.\n\nPage 9, âWhereas all three genes react rapidly to LD delaysâ, this sentence is incomplete.\n\nPage 9, bottom of the page, when discussing the effects of HFD, consider discussing whether the blunting of the feeding rhythm (which may be due to non-circadian factors, such as different dynamics of metabolism and saturation by fat vs. carbohydrates) may be the cause of the blunted clock gene rhythms, e.g., in adipose tissue and liver.\n\nPage 10, when discussing intra-cellular misalignment, please discuss to what degree the different speeds of re-entrainment following a shift between different clock genes may be due to specific clock genes acting as immediate early genes in response to the specific zeitgeber(s), which may also show transient changes in their levels in acute response to the zeitgeber.\n\nPage 10, as part of therapeutic approaches and future directions, you may want to briefly discuss timing of behavior as a therapeutic approach, e.g., food timing with promising experimental human studies including those addressing misalignment. You may also want to briefly mention the broader relevance for the timing of therapy/treatment, such as for surgery (Montaigne et. al. (2018)1).\n\nPage 10, âIt will be important to understand the mechanismsâŠâ, this paragraph describes approaches to follow cell-specific molecular rhythms under various conditions. Consider discussing experimental approaches to test whether misalignment between tissues contributes to negative health consequences.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "9455",
"date": "04 Apr 2023",
"name": "Isabel Heyde",
"role": "Author Response",
"response": "This is a timely, comprehensive, and well-written review on circadian misalignment at different organismal levels and health consequences, with a primary review of animal experimental work supplemented with human work. The claims are well supported by references to original work. My comments are mostly minor. Comment: While this is done in many cases, it is important to specify the model and tissue. This is especially important because statements about timing of peaks and troughs may be opposite between nocturnal vs. diurnal mammals, and between SCN vs. extra-SCN oscillators. For example, statements such as \"circadian locomotor output cycles kaput (CLOCK) heterodimers bind to E-box promotor elements in period1â3 (Per1â3) and cryptochrome1/2 (Cry1/2) genes inducing their transcription in the morning\" need to indicate the model and tissue. Please do this throughout the manuscript. Response: We thank the reviewer for this comment and agree that experimental model and tissue type will largely influence circadian parameters of clock genes and have therefore indicated specific model and tissue where needed. Comment: Figure 2, consider moving \"Inter-tissue desynchrony\" label to be with the third panel that is showing inter-tissue misalignment. Do you need the label âIntra-tissue desynchronyâ? This seems to refer to the same concept as âCellular desynchronyâ, i.e., misalignment between cells in the same tissue. Consider using the same terminology throughout the manuscript, and the term \"inter-cell\" to clarify that it is distinct from the intra-cellular/molecular misalignment. Response: We have relabelled \"Inter-tissue desynchrony\" for third panel in Figure 2 as suggested. With regard to âIntra-tissue desynchronyâ, we prefer to keep the label as well as the corresponding reference in the text since we want to emphasize that inter-cell and molecular desynchrony both can be grouped under this umbrella term. We changed âCellular desynchronyâ to \"Inter-cell desynchrony\" to avoid confusion. Comment: For future directions, it will be helpful for the readers to lay out what type of studies and approaches will be most valuable to clarify the knowledge gaps. This is also true for the statement on page 9, âHowever, more studies investigating the effect of phase incoherencesâŠare neededâŠâ. Response: We now elaborate on these in the last paragraph of the inter-tissue synchrony chapter and provide more detailed ideas for future directions. Please also see our response to the last comment for more details. Comment: Introduction, first paragraph, replace \"period length\" with \"period\" and consider adding \"(cycle length)\" after \"period\". Introduction, second paragraph, do the authors mean \"prevalence\" instead of \"incidence\"? Response: We thank the reviewer for these comments and changed the text accordingly. We changed the phrase from 'period length' to 'period (cycle length)' in the text. The cited studies investigated the associations between shift work/circadian disruption and the health outcome and, thereby, explored the 'incidence' so we replaced the word 'prevalence'. Comment: Introduction, second paragraph, âPossibly, this may be provoked by light-mediated resetting of the SCNâŠâ, clarify the rationale why SCN resetting, or melatonin suppression, would increase the risk for obesity or metabolic syndrome. Response: We added some more information. Briefly, LAN dampens clock gene rhythms in the SCN and may shift food intake patterns. Furthermore, we highlighted the role of melatonin in insulin biosynthesis and the regulation of leptin rhythms. Comment: Introduction, second paragraph, âShift work is believed to affectâŠâ, the phase coherence doesn't need to be between two or more circadian rhythms but could also be between a circadian and a diurnal rhythm such as between the melatonin rhythm and the sleep/wake cycle or light/dark cycle. Response: We thank the reviewer for this thoughtful comment. Indeed, phase coherence can be disturbed between two or more circadian rhythms but, as pointed out, also between circadian and diurnal rhythms. We added this in the revised manuscript. Comment: Page 4, âHowever, hepatocyte clocks in SCN ablated miceâŠâ, why would this show that peripheral tissue clocks are coupled? An alternative explanation could be that the feeding rhythm itself synchronizes hepatocytes, such that they are aligned even if they are not coupled among each other. Even though this alternative is recognized in the subsequent sentence, it will be helpful to acknowledge this in the first statement. Response: We completely agree that the aligned circadian oscillations in hepatocytes do not necessarily imply the presence of a coupling mechanism. The feeding rhythm plays a major role. Therefore, we have rephrased the text to make this clear. However, the study of Guenther et al. suggests that there is a weak coupling in peripheral oscillators so it cannot be ruled out that coupling contributes to the results observed by Saini et al..   Comment: Page 4, âWithin each rhythmic cell, the molecular clock has a normal phase distributionâ, consider rephrasing that ââŠthe components of the molecular clock have a phase distribution.â Response: We thank the reviewer for this valuable comment. We rephrased this sentence. Comment: Page 6, âAll of the above described real-life situations result in chronodisruption which promotes metabolic impairments but also increases the prevalence for major depression, cardiovascular disease, autoimmune disorders, and certain cancersâ, most of these cited studies are observational studies where other factors may also be involved, besides chronodisruption, that cause the increased disease risk, such as differences in socioeconomic status and lifestyle which will be important to acknowledge and that therefore experimental studies are needed to directly test cause-and-effect relationships with risk factors. Response: We agree that many other factors may promote the development of the listed diseases which were not mentioned in the original manuscript. Therefore, we included them in a brief explanation.  Comment: Page 9, âWhereas all three genes react rapidly to LD delaysâ, this sentence is incomplete. Response: Thank you for pointing this out. We corrected this in the revised version. Comment: Page 9, bottom of the page, when discussing the effects of HFD, consider discussing whether the blunting of the feeding rhythm (which may be due to non-circadian factors, such as different dynamics of metabolism and saturation by fat vs. carbohydrates) may be the cause of the blunted clock gene rhythms, e.g., in adipose tissue and liver. Response: We agree that dampening of clock genes may be an effect of overall dampening of feeding rhythms and have included this speculation as a brief statement.  Comment: Page 10, when discussing intra-cellular misalignment, please discuss to what degree the different speeds of re-entrainment following a shift between different clock genes may be due to specific clock genes acting as immediate early genes in response to the specific zeitgeber(s), which may also show transient changes in their levels in acute response to the zeitgeber. Response: We briefly discuss the re-entrainment speed of particular clock genes upon photic stimulation in the SCN and point out that this may partly explain the different speeds in re-entrainment seen in distinct tissues. Comment: Page 10, as part of therapeutic approaches and future directions, you may want to briefly discuss timing of behavior as a therapeutic approach, e.g., food timing with promising experimental human studies including those addressing misalignment. You may also want to briefly mention the broader relevance for the timing of therapy/treatment, such as for surgery (Montaigne et. al. (2018)1). Response: We agree that time-restricted eating is an important tool to modulate the clock and improve metabolism and, therefore, added some information on the potential of time-of-eating as a therapeutic approach. Comment: Page 10, âIt will be important to understand the mechanismsâŠâ, this paragraph describes approaches to follow cell-specific molecular rhythms under various conditions. Consider discussing experimental approaches to test whether misalignment between tissues contributes to negative health consequences. Response: We discuss the use of chimeric mice inheriting slow and normal (24-hour) clocks in distinct tissues. Furthermore, it is not known yet whether tissue-specific clock KO mice suffer from inter-tissue desynchrony which may promote/evoke the observed phenotypes. Established forced desynchrony paradigms may be useful to study the effects of inter-tissue desynchrony on health in mice. Unfortunately, in humans we so far are lacking good biomarkers for peripheral tissue clocks. We point out the need for those in the paragraph and the necessity to study desynchrony in different genders, ages, ethnicities and lifestyles to evaluate the effect on health."
}
]
},
{
"id": "161398",
"date": "23 Feb 2023",
"name": "Andrew N. Coogan",
"expertise": [
"Reviewer Expertise Chronobiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a nicely written and timely review of the literature on circadian desynchrony, its mechanisms and its impacts on health. It provides a multi-level framework through which circadian desynchrony may be considered, and as such represents a very useful addition to the literature for researchers in chronobiology and adjacent areas.\nI have only minor comments and suggestions that could be considered for an update of the manuscript.\nI would suggest adding a brief discussion of the very recently published studies on multi-tissue rhythms in humans (Wucher et al. (2023)1 and Talamanca et al. (2023)2). I appreciate that these papers have been published since the posting of the current version, but they are so pertinent to the concept of the review that an update might be warranted.\n\nIn general, whilst discussing the epidemiological evidence concerning shift work as a risk factor for various chronic diseases, I would note that the literature is more mixed than presented, and that findings of associations between shift work and various chronic health outcomes tend to be far from ubiquitous. I would also note that the exposure to shift work probably consists of elements other than circadian desynchrony/light-at-night exposure (such as shorter sleep duration, poorer sleep quality, increased alcohol and nicotine use as coping mechanisms, poorer diet quality). I think it would be helpful as such to contextualize the discussion of shift work and circadian desynchrony a little more.\n\nI suggest that healthy physiological aging is a \"condition\" that may also warrant attention with reference to circadian desynchrony. There are a number of studies demonstrating that circadian desynchrony appears to be associated with \"healthy\" aging. Further, as many of the chronic conditions discussed in relation to desynchrony are age-related, it is important to consider circadian changes in the pre-morbid state. As such, when discussing evidence from disease models for desynchrony, I would find it useful if mention was made of the ages of the animals examined.\n\nFor Figure 2 and its associated discussion, I would find it useful if the \"misaligned zeitgebers\" were indicated as both misaligned with reference to each other (e.g. meal timing from light/dark cycles), and also misaligned with the internal circadian phases. I think this is implicit in the discussion, but suggest that it would be helpful to make these considerations explicit.\n\nMy preference would be for the consideration of social jetlag to be given more prominence, given its near ubiquitous nature at the population level as an everyday circadian desynchrony. I think it would also be helpful for the reader if indications of the non-trivial magnitude of social jetlag could be indicated in the manuscript.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "9456",
"date": "04 Apr 2023",
"name": "Isabel Heyde",
"role": "Author Response",
"response": "This is a nicely written and timely review of the literature on circadian desynchrony, its mechanisms and its impacts on health. It provides a multi-level framework through which circadian desynchrony may be considered, and as such represents a very useful addition to the literature for researchers in chronobiology and adjacent areas. I have only minor comments and suggestions that could be considered for an update of the manuscript. Comment: I would suggest adding a brief discussion of the very recently published studies on multi-tissue rhythms in humans (Wucher et al. (2023)1 and Talamanca et al. (2023)2). I appreciate that these papers have been published since the posting of the current version, but they are so pertinent to the concept of the review that an update might be warranted. Response: We agree that these references serve as important recent additions to the field. We included these in the organization of the mammalian circadian clock system in the sections where we are highlighting the phase relationship between tissues and genes. However, we feel the need to point out the limitation of the study design while acknowledging the fact that the GTEx data set is so far the best data set available. The main limitation we find with this data set is that data were obtained from hospitalised patients which indirectly points out that there might be a high possibility of the patients being in a chronodisrupted health condition.  Comment: In general, whilst discussing the epidemiological evidence concerning shift work as a risk factor for various chronic diseases, I would note that the literature is more mixed than presented, and that findings of associations between shift work and various chronic health outcomes tend to be far from ubiquitous. I would also note that the exposure to shift work probably consists of elements other than circadian desynchrony/light-at-night exposure (such as shorter sleep duration, poorer sleep quality, increased alcohol and nicotine use as coping mechanisms, poorer diet quality). I think it would be helpful as such to contextualize the discussion of shift work and circadian desynchrony a little more. Response: We added studies showing that shift work does not necessarily increase the risk for distinct diseases. Furthermore, we explained that the exposure to shift work does not only include circadian desynchrony/light-at-night exposure but also has a negative impact on the sleep quality and related coping mechanisms. Comment: I suggest that healthy physiological aging is a \"condition\" that may also warrant attention with reference to circadian desynchrony. There are a number of studies demonstrating that circadian desynchrony appears to be associated with \"healthy\" aging. Further, as many of the chronic conditions discussed in relation to desynchrony are age-related, it is important to consider circadian changes in the pre-morbid state. As such, when discussing evidence from disease models for desynchrony, I would find it useful if mention was made of the ages of the animals examined. Response: We thank the reviewer for this comment. We agree and briefly discuss the relationship between aging and circadian desynchrony and highlight the point that considering age of experimental animals should be given more importance. We do not explicitly mention the age of experimental subjects in every referred study. However, we do make a statement, before starting the main subject chapters, mentioning that all the cited studies were conducted in young adults of the respective model if not stated otherwise.  Comment: For Figure 2 and its associated discussion, I would find it useful if the \"misaligned zeitgebers\" were indicated as both misaligned with reference to each other (e.g. meal timing from light/dark cycles), and also misaligned with the internal circadian phases. I think this is implicit in the discussion, but suggest that it would be helpful to make these considerations explicit. Response: We agree that this is an important point that was missed in the figure representation and have now corrected the figure by displaying different panels for both types of \"misaligned zeitgebersâ. Comment: My preference would be for the consideration of social jetlag to be given more prominence, given its near ubiquitous nature at the population level as an everyday circadian desynchrony. I think it would also be helpful for the reader if indications of the non-trivial magnitude of social jetlag could be indicated in the manuscript. Response: Impact of social jetlag in everyday life was not given prominence in the original manuscript. We agree that this is a very valuable addition and now introduce the social jetlag concept in the introduction as well as discuss its effects at the population level in the following chapters."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1323
|
https://f1000research.com/articles/10-1266/v1
|
09 Dec 21
|
{
"type": "Research Article",
"title": "Quantitative image analysis in COVID-19 acute respiratory distress syndrome: a cohort observational study.",
"authors": [
"Tamas Dolinay",
"Dale Jun",
"Abigail Maller",
"Augustine Chung",
"Brandon Grimes",
"Lillian Hsu",
"David Nelson",
"Bianca Villagas",
"Grace Hyun J Kim",
"Jonathan Goldin",
"Dale Jun",
"Abigail Maller",
"Augustine Chung",
"Brandon Grimes",
"Lillian Hsu",
"David Nelson",
"Bianca Villagas",
"Grace Hyun J Kim",
"Jonathan Goldin"
],
"abstract": "Background Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury commonly associated with pneumonia, including coronavirus disease-19 (COVID-19). The resultant effect can be persistent lung damage, but its extent is not known. We used quantitative high resolution computed tomography (QHR-CT) lung scans to radiographically characterize the lung damage in COVID-19 ARDS (CARDS) survivors. Methods Patients with CARDS (N=20) underwent QHR-CT lung scans 60 to 90 days after initial diagnosis, while hospitalized at a long-term acute care hospital (LTACH). QHR-CT assessed for mixed disease (QMD), ground glass opacities (QGGO), consolidation (QCON) and normal lung tissue (QNL). QMD was correlated with respiratory support on admission, tracheostomy decannulation and supplementary oxygen need on discharge. Results Sixteen patients arrived with tracheostomy requiring invasive mechanical ventilation. Four patients arrived on nasal oxygen support. Of the patients included in this study 10 had the tracheostomy cannula removed, four remained on invasive ventilation, and two died. QHR-CT showed 45% QMD, 28.1% QGGO, 3.0% QCON and QNL=23.9%. Patients with mandatory mechanical ventilation had the highest proportion of QMD when compared to no mechanical ventilation. There was no correlation between QMD and tracheostomy decannulation or need for supplementary oxygen at discharge. Conclusions Our data shows severe ongoing lung injury in patients with CARDS, beyond what is usually expected in ARDS. In this severely ill population, the extent of mixed disease correlates with mechanical ventilation, signaling formation of interstitial lung disease. QHR-CT analysis can be useful in the post-acute setting to evaluate for interstitial changes in ARDS.",
"keywords": [
"acute respiratory distress syndrome",
"COVID-19",
"quantitative high-resolution lung CT scanning"
],
"content": "Introduction\n\nAcute respiratory distress syndrome (ARDS) is a severe form of lung injury requiring intensive care unit (ICU) hospitalization. The etiology of ARDS is broad, but approximately 40% of cases are complications of respiratory infections.1 Despite modern intensive care the mortality of ARDS remains above 30%2,3 and international statistics show that ARDS is responsible for approximately 10% of all ICU admissions.3 During the 2020-2021 coronavirus disease (COVID)-19 pandemic, ARDS emerged as a feared complication of COVID-19 pneumonia driving ICU hospitalizations and mortality.4,5 Clinical data also suggest that recovery from COVID-19-associated ARDS (CARDS) is prolonged requiring ongoing respiratory support beyond what is traditionally seen in ICU care.6,7\n\nHigh resolution-computer tomography (HR-CT) scans with quantitative analysis (QHR-CT) have been widely used to study the details of the lung parenchyma8,9 and have been beneficial in tracking the progression of interstitial lung disease.10 In ARDS, the use of CT analysis was initially hindered by concerns over transportation of the critically ill. However with portable and faster CT scanners available in routine clinical care, HR-CTs have proved its usefulness in determining alveolar damage and edema formation.11,12 Less is known regarding its utility in following the clinical course of the disease as lung infiltrates seen in the acute phase of ARDS usually resolve.13 In spite of this, in a minority of patients, pulmonary fibrosis may develop which has been associated with poor outcome.14 Recently McGroder et al. and in a separate analysis Gonzalez et al. reported persistent lung infiltrates and fibrosis-like changes 3-4 months after severe COVID pneumonia.6,7 This may suggest that persistent lung changes in ARDS are more common than initially reported.\n\nIn our study, we examined the extent of persistent lung changes in CARDS survivors requiring continued hospitalization. Our findings suggest ongoing lung damage, which may give rise to pulmonary fibrosis.\n\n\nMethods\n\nOur study was approved by the Western Institutional Review Board (IRB) protocol ID #20210635. In this single center observational cohort study, patients admitted to Barlow Respiratory Hospital (BRH) with a diagnosis of CARDS were considered for enrollment. To avoid selection bias, we approached consecutively admitted patients to participate in the study. BRH is a non-profit long-term acute care hospital (LTACH) serving the greater Los Angeles area. Patients are transferred to BRH for ongoing respiratory care from short-term acute care hospitals (STACH). CARDS diagnosis was made during STACH hospitalization based on the following criteria: 1. at least one positive COVID-19 PCR test on admission to STACH, 2. new bilateral lung infiltrates in the past seven days on chest imaging not attributed to pulmonary edema alone, 3. requirement for invasive or non-invasive mechanical ventilation with at least 5 cm H2O positive pressure, 4. ratio of partial arterial oxygen pressure (PaO2) and fraction of inspired oxygen (FiO2) <300.\n\nTo participate in the study, informed written consent was obtained directly from the patient by the investigators. In case, the patient was not directly consentable, per the IRB recommendations, consent was obtained from the patientâs power of attorney. We enrolled 25 patients in our study between February and July 2021. Patients were consented to undergo lung QHR-CT during BRH admission. From the 25 enrolled patients, 20 completed the CT scans and their demographic and clinical data was collected by chart review. Data was entered in a password protected database. We did not record data of the patients, who did not complete the CT scans to avoid bias from an incomplete dataset.\n\nThe original QHR-CT scans and database contain sensitive patient information and is not publicly available for review per IRB guidelines. We created a deidentified database to share with the readers, which contains all pertinent patient demographics, clinical data, QHR-CT scan scores and volume measurements. This database is uploaded in a public database.25\n\nPatientsâ age, gender, race, ethnicity, STACH admission date, LTACH admission date, CT scan date, premorbid medical condition, tracheostomy status on admission, presence or absence of mechanical ventilation and mechanical ventilation mode were collected at the time of LTACH admission. The following premorbid conditions were considered: bacterial pneumonia, pneumothorax, acute or chronic kidney disease, hypertension, diabetes mellitus, deep vein thrombosis (DVT), pulmonary embolism (PE), heart failure, coronary artery disease (CAD), cerebrovascular accident (CVA), obesity with body mass index greater than 30 and pulmonary fibrosis. During LTACH stay data was collected for inpatient death, need for continued mechanical ventilation, on tracheostomy decannulation status and fraction of inspired oxygen (FiO2) on discharge.\n\nHR-CT lung scans were performed using a General Electric BrightSpeed 16 slice CT scanner (Model # 5128609-2, General Electric Health Corporation, Chicago, IL, USA) with 2mm cuts at BRH. QHR-CT analysis was performed by University of California Los Angeles (UCLA) Radiology department. Quantitative scores were measures for four distinct radiological patterns: 1. ground glass opacity, (QGGO), 2. mixed diseases (QMD), 3. consolidation (QCON) and 4. normal lung (QNL). The sum of three abnormal lung tissue scores was named the quantitative total lung diseases (QTLD). We applied the domain adaptation for calculating quantitative COVID-19 scores from HR-CT images. The source data and technique were adapted from the previously developed algorithm for diffuse lung disease15,16 and the target data was HR-CT images containing consolidation.17 The final model was reviewed and visually confirmed using an independent COVID-19 cohort at UCLA. Ground glass opacities usually represent acute inflammatory processes, mixed disease is commonly a radiological presentation of interstitial lung disease (ILD) and consolidation is frequently associated with pulmonary infection.18\n\nBased on the need for mechanical ventilation at LTACH admission, three groups of patients were created: 1. mandatory mode mechanical ventilation via tracheostomy (MV), 2. spontaneous mode mechanical ventilation via tracheostomy (SV) and no need for mechanical ventilation (NV). MV included volume control ventilation, pressure control ventilation, and synchronized intermittent mandatory ventilation. SV included pressure support ventilation.\n\nPatient characteristics and mechanical ventilation data were expressed as a percentage of total. Age and FiO2 were expressed as mean± standard deviation (SD). The lung disease score was expressed as proportion to total lung capacity (TLC) ± SD. The extent of mixed disease (QMD) was expressed as QMD/TLC± standard error (SE) and QMD/QTLD± SE. QMD was correlated with the presence of mechanical ventilation on admission, tracheostomy decannulation. Statistical analysis was performed using non-parametric Kruskal-Wallis equality-of-population rank test with Bonferroni correction for the multiple comparisons. p < 0.05 was considered statistically significant. Correlation coefficient was calculated between the discharge FiO2, QMD and QMD/QTLD and data is shown with 95% confidence interval (95%CI). Stata 14.1 software (College Station, Texas 77845 USA, RRID:SCR_012763) was used for statistical analysis.\n\n\nResults\n\nWe analyzed the CT images of 8 female and 12 male patients. The mean age was 61.2 years. There were 2 Asian, 1 American Indian/Native Alaskan, 3 African American, 6 Latino and 8 White patients in our cohort. Twelve patients were Non-Hispanic and 8 patients were Hispanic. At the time of LTACH admission 16 patients had tracheostomy and required invasive mechanical ventilation, 2 patients arrived on high flow oxygen support and 2 patients needed low flow oxygen. Of the 20 patients, 16 patients had secondary bacterial, 7 patients had pneumothorax secondary to mechanical ventilation, 9 patients had renal disease, 12 had hypertension, 12 had diabetes mellitus, 6 had DVT, 2 had PE, 3 patients suffered from heart disease, 3 had recent CVA, 9 were obese and none of the patients had a prior diagnosis of pulmonary fibrosis. Demographics and premorbid conditions are shown in Table 1.\n\nSpecifics of airway management, mechanical ventilation and liberation from mechanical ventilation are listed in Table 2 and can also be found in the online database. Sixteen patients required invasive mechanical ventilation via tracheostomy on LTACH admission. Of those 13 arrived with MV and 3 with SV. Four patients needed supplementary oxygen via nasal cannula, 2 with high flow and 2 with low flow (less than 10 liters per minute) systems. Twelve patients were liberated from mechanical ventilation and 10 patients had the tracheostomy cannula removed (decannulated) before LTACH discharge. Two patients needed continued MV on discharge. Two patients died during the LTACH stay. No patients required resumption of mechanical ventilation. Five patients were discharged on room air. The average supplementary FiO2 on discharge was 27.6% (SD = 6.8).\n\nQuantitative lung injury scores correlate with disease severity on admission\n\nQHR-CT was performed between two and three months post diagnosis of CARDS. Average TLC was low 2175.3 ml (SD = 574.1). There was significant persistent lung damage with QTLD = 76.1% (SD = 12.1) of TLC. The majority of lung pathology was QMD with 45.0% (SD = 16.1) of TLC. QGGO was 28.1% (SD = 9.6), QCON was 3.0% (SD = 3.7) with little normal lung tissue remaining QNL = 23.9% (SD = 12.1). The distribution of lung disease was equal in both lungs (data not shown). Table 3 shows the extent of lung disease in relation to TLC. Figure 1A shows an example of the distribution of lung disease on CT scan and Figure 1B depicts the patterns of lung abnormalities in color coded fashion.\n\nA. Horizontal high resolution (HR)-CT lung cut of a selected COVID-19 ARDS (CARDS) patient. The patient is a 61-year-old male with mandatory mechanical ventilation on arrival to the long-term-acute care facility.\n\nB. Color coded quantitative HR-CT (QHR-CT) analysis in CARDS. Quantitative scores were 19.1% mixed disease (QMD), 25.1% of ground glass opacities (QGGO) and 3.4% consolidation (QCON). The ratio of QMD to quantitative total lung diseases (QTLD) was 40.1%. Red and blue = mixed disease (QMD), yellow and cyan = ground glass opacities (QGGO), peach = consolidation (QCON).\n\nThe extent of QMD was correlated with admission use of respiratory support (Table 4). Patients with MV and SV had more QMD% than their NV counterparts. When QMD/QTLD ratio was calculated, MV patients had significantly higher ratio than NV patients (p < 0.0127). There was no correlation between the extent of QMD and tracheostomy decannulation or need for supplementary oxygen on discharge (Table 4).\n\n* Represents significant difference between MV and NV, p = 0.0127, Kruskal-Wallis test; SE: standard error.\n\n\nDiscussion\n\nModern ICU care significantly improved the immediate survival of ARDS, but little is known about the long-term respiratory complications of the disease.3,19 Herridge et al. reported that 20% of ARDS survivors have abnormal chest imaging at one year20 and more recently Burnham et al. showed that 25% ARDS survivors may have ILD at six months.21 The etiology of persistent interstitial lung changes post ARDS is not well understood, but it has been associated with poor quality of life.14,21 During the COVID-19 pandemic, ARDS cases soared5 and preliminary studies suggest that 30-40% of critically ill COVID-19 patients have persistent lung changes.6,7 It has been long speculated that a major factor for the development in ILD in ARDS is invasive positive pressure mechanical ventilation (commonly abbreviated mechanical ventilation).22 Although lifesaving, mechanical ventilation has been associated with increased rates of pulmonary fibrosis post ARDS.14 Recently McGroder et al. showed fibrosis-like radiographic changes in 72% patients receiving mechanical ventilation compared to 20% of non-ventilated COVID-19 patients.6 The etiology of fibrosis is unclear but our imaging analysis in agreement with other studies that describe a non-specific post-inflammatory origin.7,14 All together this data suggests that survivors of CARDS maybe prone to ILD and pulmonary fibrosis thus, requiring long-term monitoring.23\n\nIn our study we performed QHR-CT lung analysis of CARDS survivors, who require ongoing respiratory care two to three months after the initial diagnosis. LTACH patients represent a unique population of the chronically critically ill with significant morbidity and mortality. We have previously reported that 80% of COVID-19 patients requiring LTACH admission have tracheostomy and 51% are receiving mechanical ventilation.24 In this severely ill population, it is difficult to apply lung function testing and quality of life questionnaires to assess respiratory status. QHR-CT has been useful in following patients with ILD and can detect disease progression.10 HR-CT can be relatively easily performed in non-cooperative patients and quantitative analysis provides insight to the ongoing lung disease. We found that in our population of CARDS patients, there was significant lung disease involving, on average, 76% of the lungs. The most significant form of lung changes (45%) were consistent with mixed disease, which is a combination of reticulation and traction bronchiectasis, suggesting ILD. ILD changes usually result in permanent scaring and can lead to pulmonary fibrosis. In comparison, ground glass opacities and consolidation seen with acute inflammation and infections usually resolve. These findings suggest that ILD is more common in CARDS than other forms of ARDS. We also observed that patients who did not require mechanical ventilation on admission, had less ILD, which is consistent with findings of McGroder et al.6 However, we did not find association between the extent of mixed disease and tracheostomy decannulation or need for supplementary oxygen at discharge. This data signals that the cession of mechanical ventilation or lower oxygen supplementation will not reverse the damage that has already occurred.\n\nOur study has several strengths: 1. it studies a chronically critically ill population, in which the outcomes of ARDS are not known, 2. it shows that QHR-CT can be easily used to study lung disease in a population where traditional respiratory tests are difficult to perform, 3. it adds to our understanding of ILD development post ARDS. However, our study also has limitations: 1. it studies a small group of CARDS patients in a single hospital in Los Angeles, California, which may limit generalizability; 2. CARDS is a complex disease and our limited dataset allowed only the analysis of a select number of respiratory parameters, which may not have taken into account other possible confounders in our analysis; 3. QHR-CT technology is not specific to ARDS and some of the unique pathologic and radiographic changes of this disease may have been missed; 4. we did not perform serial imaging and we do not know, if the observed mixed disease, will progress with time; 5. lastly, we used HR-CT technology which results in radiation exposure.\n\n\nConclusions\n\nIn conclusion, our study suggests that lung disease is highly prevalent in CARDS two to three months after the initial infection. ILD is the most prominent findings on imaging, which may result in progression to fibrotic disease. We recommend following CARDS patients with HR-CT beyond the acute care setting to evaluate for the development of ILD.\n\n\nData availability\n\nOSF: CT in COVID https://doi.org/10.17605/OSF.IO/S2FXN.25\n\nThe project contains the following underlying data:\n\n⢠CT database-deidentified_final_with_CT_scores_11-18-21.xlsx\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nConsent statement\n\nInformed written consent was obtained directly from the patient by the investigators. In case, the patient was not directly contactable, per the IRB recommendations, consent was obtained from the patientâs power of attorney.",
"appendix": "References\n\nRubenfeld GD, Caldwell E, Peabody E, et al.: Incidence and outcomes of acute lung injury. N Engl J Med. 2005; 353(16): 1685â1693. Publisher Full Text\n\nCochi SE, Kempker JA, Annangi S, et al.: Mortality Trends of Acute Respiratory Distress Syndrome in the United States from 1999 to 2013. Ann Am Thorac Soc. 2016; 13(10): 1742â1751. PubMed Abstract | Publisher Full Text\n\nBellani G, Laffey JG, Pham T, et al.: Epidemiology, Patterns of Care, and Mortality for Patients With Acute Respiratory Distress Syndrome in Intensive Care Units in 50 Countries. JAMA. 2016; 315(8): 788â800. PubMed Abstract | Publisher Full Text\n\nYang X, Yu Y, Xu J, et al.: Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. Lancet Respir Med. 2020; 8(5): 475â481. PubMed Abstract | Publisher Full Text\n\nMyers LC, Parodi SM, Escobar GJ, et al.: Characteristics of Hospitalized Adults With COVID-19 in an Integrated Health Care System in California. JAMA. 2020; 323(21): 2195â2198. PubMed Abstract | Publisher Full Text\n\nMcGroder CF, Zhang D, Choudhury MA, et al.: Pulmonary fibrosis 4 months after COVID-19 is associated with severity of illness and blood leucocyte telomere length. Thorax. 2021; 76: 1242â1245. PubMed Abstract | Publisher Full Text\n\nGonzalez J, Benitez ID, Carmona P, et al.: Pulmonary Function and Radiologic Features in Survivors of Critical COVID-19: A 3-Month Prospective Cohort. Chest. 2021; 160(1): 187â198. PubMed Abstract | Publisher Full Text\n\nRobbie H, Wells AU, Jacob J, et al.: Visual and Automated CT Measurements of Lung Volume Loss in Idiopathic Pulmonary Fibrosis. AJR Am J Roentgenol. 2019; 213(2): 318â324. PubMed Abstract | Publisher Full Text\n\nElicker BM, Kallianos KG, Henry TS: The role of high-resolution computed tomography in the follow-up of diffuse lung disease: Number 2 in the Series âRadiologyâ Edited by Nicola Sverzellati and Sujal Desai. Eur Respir Rev. 2017; 26(144): 170008. PubMed Abstract | Publisher Full Text\n\nGoldin JG, Kim GHJ, Tseng CH, et al.: Longitudinal Changes in Quantitative Interstitial Lung Disease on Computed Tomography after Immunosuppression in the Scleroderma Lung Study II. Ann Am Thorac Soc. 2018; 15(11): 1286â1295. PubMed Abstract | Publisher Full Text\n\nGaliatsou E, Kostanti E, Svarna E, et al.: Prone position augments recruitment and prevents alveolar overinflation in acute lung injury. Am J Respir Crit Care Med. 2006; 174(2): 187â197. PubMed Abstract | Publisher Full Text\n\nLeiser P, Kirschning T, Weiss C, et al.: A quantitative CT parameter for the assessment of pulmonary oedema in patients with acute respiratory distress syndrome. PLoS One. 2020; 15(11): e0241590. PubMed Abstract | Publisher Full Text\n\nMasclans JR, Roca O, Munoz X, et al.: Quality of life, pulmonary function, and tomographic scan abnormalities after ARDS. Chest. 2011; 139(6): 1340â1346. PubMed Abstract | Publisher Full Text\n\nCabrera-Benitez NE, Laffey JG, Parotto M, et al.: Mechanical ventilation-associated lung fibrosis in acute respiratory distress syndrome: a significant contributor to poor outcome. Anesthesiology. 2014; 121(1): 189â198. PubMed Abstract | Publisher Full Text\n\nKim HG, Tashkin DP, Clements PJ, et al.: A computer-aided diagnosis system for quantitative scoring of extent of lung fibrosis in scleroderma patients. Clin Exp Rheumatol. 2010; 28(5 Suppl 62): S26âS35. PubMed Abstract\n\nKim HJ, Brown MS, Chong D, et al.: Comparison of the quantitative CT imaging biomarkers of idiopathic pulmonary fibrosis at baseline and early change with an interval of 7 months. Acad Radiol. 2015; 22(1): 70â80. PubMed Abstract | Publisher Full Text\n\nAllwood BW, Maasdorp E, Kim GJ, et al.: Transition from Restrictive to Obstructive Lung Function Impairment During Treatment and Follow-Up of Active Tuberculosis. Int J Chron Obstruct Pulmon Dis. 2020; 15: 1039â1047. PubMed Abstract | Publisher Full Text\n\nParekh M, Donuru A, Balasubramanya R, et al.: Review of the Chest CT Differential Diagnosis of Ground-Glass Opacities in the COVID Era. Radiology. 2020; 297(3): E289âE302. PubMed Abstract | Publisher Full Text\n\nBurnham EL, Janssen WJ, Riches DW, et al.: The fibroproliferative response in acute respiratory distress syndrome: mechanisms and clinical significance. Eur Respir J. 2014; 43(1): 276â285. PubMed Abstract | Publisher Full Text\n\nHerridge MS, Cheung AM, Tansey CM, et al.: One-year outcomes in survivors of the acute respiratory distress syndrome. N Engl J Med. 2003; 348(8): 683â693. PubMed Abstract | Publisher Full Text\n\nBurnham EL, Hyzy RC, Paine R 3rd, et al.: Chest CT features are associated with poorer quality of life in acute lung injury survivors. Crit Care Med. 2013; 41(2): 445â456. PubMed Abstract | Publisher Full Text\n\nMarshall R, Bellingan G, Laurent G: The acute respiratory distress syndrome: fibrosis in the fast lane. Thorax. 1998; 53(10): 815â817. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGeorge PM, Barratt SL, Condliffe R, et al.: Respiratory follow-up of patients with COVID-19 pneumonia. Thorax. 2020; 75(11): 1009â1016. PubMed Abstract | Publisher Full Text\n\nHassenpflug MS, Jun D, Nelson DR, et al.: Post-COVID recovery: characteristics of chronically critically ill patients admitted to a long-term acute care hospital. F1000Res. 2020; 9: 1241. Publisher Full Text\n\nDolinay T, Jun D: CT in COVID. OSF. 2021. Publisher Full Text"
}
|
[
{
"id": "135370",
"date": "03 May 2022",
"name": "Jeffrey Haspel",
"expertise": [
"Reviewer Expertise Pulmonary and Critical Care. Circadian biology. Inflammation"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere, Dolinay and colleagues report the prevalence of radiographic abnormalities in a cohort of COVID-19 survivors admitted to their LTACH. They note a high prevalence of CT abnormalities 2-3 months post COVID, consistent with ILD, and based on this suggest the need for long term radiographic follow up.\n\nOverall, this is an interesting report that, combined with reports sure to follow from other centers, will be useful in clarifying the natural history of severe COVID-19.\n\nMy comments are minor:\nSince TLC is reported as \"low\" based on CT, it would be helpful to supply the %predicted TLC.\n\nSince the use of Kruskal Wallis suggests the data presented are non-parametric it is probably more correct to report IQR rather than SD/SE in Table 3 and in the text (SD/SE usually refer to normally distributed data).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "8271",
"date": "24 May 2022",
"name": "Tamas Dolinay",
"role": "Author Response",
"response": "We would like to thank the reviewer for his useful comments. Please see below our point-by-point response: 1. In the revised manuscript, we included the percent of predicted total lung capacity (% of TLC) for our patients (N=20) with standard deviation (SD). 2. We agree with the reviewer that presenting the data with interquartile ranges (IQR) is more representative when used with non-parametric testing. We now show IQR for both Table 3 and 4."
}
]
}
] | 1
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https://f1000research.com/articles/10-1266
|
https://f1000research.com/articles/11-388/v1
|
04 Apr 22
|
{
"type": "Case Report",
"title": "Case Report: Identification of likely recurrent CEP290 mutation in a child with Joubert syndrome and cerebello-retinal-renal features.",
"authors": [
"Lidvana Spahiu",
"John A Sayer",
"Emir Behluli",
"Thomas Liehr",
"Gazmend Temaj",
"Lidvana Spahiu",
"Emir Behluli",
"Thomas Liehr",
"Gazmend Temaj"
],
"abstract": "Background. Joubert syndrome (JS) is a rare autosomal recessive ciliopathy with an estimated prevalence of 1 in 100,000. JS is characterized by hyperpnoea, hypotonia, ataxia, developmental delay and various neuropathological abnormalities in the brain including cerebellar hypoplasia and cerebellar vermis aplasia. JS can also have variable multi-organ involvement, including the retina, kidneys, liver, and musculoskeletal system. Methods and Results. Here we report a clinical description of two-year-old girl presenting with breathing difficulties, hyperechoic kidneys with loss of corticomedullary differentiation. Brain magnetic resonance imaging revealed the typical molar tooth sign consistent with a clinical diagnosis of JS and retinal examination showed severe retinal dystrophy leading to blindness. Molecular genetic analysis using whole exome sequencing and Sanger sequence confirmation demonstrated a homozygous mutation (c.5493delA, p.(A1832fs*19) in CEP290 which segregated from either parent and was consistent with the multisystem ciliopathy phenotype. This precise variant has been described previously in 2 families from the Kosovar-Albanian region suggesting this allele is a recurrent mutation in this population. Conclusions. Mutations in CEP290 lead to multisystem ciliopathy syndromes and molecular genetic diagnostics of such cases allows precise diagnosis, screening of at risk relatives and appropriate management.",
"keywords": [
"Joubert syndrome",
"CEP290",
"molecular genetics",
"ciliopathy",
"retinal dystrophy"
],
"content": "Introduction\n\nJoubert syndrome (JS, MIM 213300) is a rare heterogeneous neurodevelopmental disease, being associated with an autosomal or X-chromosomal recessive inheritance. There are at least 34 genes (OMIM PS213300) associated with different subtypes of JS (Parisi and Glass 1993). JS can present clinically with different symptoms including hypotonia progressing to ataxia, global development delay, eye movement abnormalities (nystagmus and ocular molar apraxia) and dysregulation of breathing. Other clinical features found in JS patients depends upon other systemic involvement but includes retinal dystrophy, hepatic fibrosis, polycystic kidney disease and polydactyly (Brancati et al. 2010; Parisi and Glass 1993; Romani et al. 2013; Sayer et al. 2006; Valente et al. 2013). The prevalence of JS is approximately 1 in 100,000 of live births worldwide. From the currently known JS genes, TMEM67 is the most frequent disease causing gene in European and Japanese patients (Bachmann-Gagescu et al. 2015; Kroes et al. 2016; Suzuki et al. 2016) and is one of the most frequent cause of JS with renal involvement (Fleming et al. 2017). CEP290 is also a frequent cause of JS associated with kidney phenotypes (Bachmann-Gagescu et al. 2015; Vilboux et al. 2017). Here we report a patient from Kosovo with a clinical diagnosis of JS with kidney and retinal involvement in whom we identified a homozygous frameshift mutation in CEP290.\n\n\nCase presentation\n\nThe Institutional Review Board of Department of Paediatrics, Clinical University Center, Kosovo, approved this work. The parents of the patient provided their written informed consent before clinical and laboratory examinations and for publication of the case. Blood from the patient and his healthy parents were obtained to allow DNA extraction and genetic studies. All the procedures performed in the study were in accordance with the Helsinki Declaration.\n\nThe female patient was born to a not knowingly consanguineous couple from Kosovo. The patient had three healthy siblings, two older sisters and one younger brother. During antenatal scans, ill-defined brain morphology changes were noted. Following birth, via Caesarian section at 38 weeks gestation, the birth weight was 3460 g, length 50 cm, and head circumference 35 cm. Following birth, the baby became dyspneic and cyanotic, and was treated with oxygen therapy (FiO2 30%). Externally, there were no dysmorphic signs. At age of 1 year, the child had evidence of poor growth: weight 7610 g (below 5th percentile), height 75 cm (below 25th percentile), head circumference 48 cm (below 5th percentile). At 2 years of age, the child demonstrated features of developmental delay and was unable to sit or walk. She had started mumbling at the age of 18 months but did not develop any coherent speech. She had horizontal gaze nystagmus and ptosis in right eye. Abdominal ultrasound scanning showed enlarged, hyperechoic kidneys with loss of corticomedullary differentiation, with a number of small cysts up to 12 mm in diameter. There was evidence of progressive chronic kidney disease with serum creatinine 243 ÎŒmol/L. Magnetic resonance imaging (MRI) of the head and neck suggested a Dandy Walker anomaly with no clear signs of foramina magnum obstruction and a typical âmolar tooth signâ. Eye examination revealed severe retinal dystrophy and ptosis of the right eyelid. Molecular genetic analysis using exome sequencing and Sanger sequence confirmation revealed a homozygous frameshift mutations c.5493delA; p.A1832fs*19 in CEP290 (Figure 1). This variant segregated from each parent.\n\nA. Pedigree diagram; squares, males; circles, females; shaded, affected. B. Photograph of proband aged 3 years, showing right ptosis. C. Brain MRI demonstrating molar tooth sign (arrowed). D. Abdominal MRI demonstrating bilateral enlarged kidneys. E. Sanger sequencing showing homozygous pathogenic frameshift mutation segregating from each parent. F. gnomAD frequencies of pathogenic allele CEP290 NM_25114 c.5493delA; A1832Pfs*19 and CEP290 protein showing domain structure and location of frameshift mutation which truncates C-terminal domains.\n\n\nDiscussion\n\nJS originally was described in 1968 in a Canadian French family an index patient showing malformation of the cerebellar vermis, with intellectual disability, ataxia, abnormal eye movements, and episodic hyperpnoea (Brancati et al. 2010; Joubert et al. 1969). JS has several phenotypic subtypes including âpureâ JS, JS with ocular defects, JS with renal defects, JS oculorenal disorders, JS with hepatic disorders and JS with orofaciodigital defects. Of the known 34 genes implicated in JS, 33 genes are autosomal recessive, and one is X-linked (Coene et al. 2009; Parisi 2009). The most striking clinical features for JS are episodic hyperpnoea and apnoea during the neonatal period, ocular disorders, hypotonia truncal ataxia, developmental delay and intellectual impairment (Kumar et al. 2007; Poretti et al. 2011). Here we report a patient with a molar tooth sign on brain MRI imaging with retinal and kidney involvement in whom we found using exome sequencing a homozygous frameshift mutation in CEP290 (NM_025114.4 c.5493delA; p.A1832fs*19, ClinVar https://www.ncbi.nlm.nih.gov/clinvar/variation/56739/). This variant, located in exon 29 and predicted to truncate the C-terminus of the CEP290 protein (Figure 1), has been identified in a wide spectrum of ciliopathy phenotypes from Leber congenital amaurosis (Carss et al. 2017; Cideciyan et al. 2011; Coppieters et al. 2010; Feldhaus et al. 2020), JS with renal and retinal involvement (Travaglini et al. 2009) and Meckel syndrome (Frank et al. 2008; Tallila et al. 2009) (Table 1). Interestingly, of these families with Meckel syndrome and the CEP290 c.5493delA allele, one consanguineous multiplex family and a second consanguineous family, not knowingly related to the first, were from the Kosovo-Albanian region suggesting this allele might be a recurrent or founder allele from this population.\n\n* Two consanguineous families of Kosovar-Albanian origin.\n\nIn conclusion, JS is often a multi-organ primary ciliopathy syndrome and patients should undergo a clinical and imaging diagnostic protocol to evaluate possible different abnormalities (Bachmann-Gagescu et al. 2020) and genetic testing to allow identification of the underlying cause. Our results highlight the importance of combining clinical and radiological features of JS with molecular genetic analysis to allow a precise diagnosis of JS. An accurate diagnosis helps in genetic counselling, early management of genetic disorders and offers prenatal diagnosis as an option for future pregnancies. It also allows genotype-phenotype correlations to be determined and allows new information to be gained regarding population specific disease alleles.\n\nThe original contributions presented in the study are publicly available on LOVD (Fokkema et al. 2011). This data can be found here: https://databases.lovd.nl/shared/individuals/00402012.\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nEthics statement\n\nThe Institutional Review Board of Department of Paediatrics, Clinical University Center, Kosovo, approved this work. The studies involving human participants were reviewed and approved by North-East Newcastle and North Tyneside 1 Research Ethics Committee (18/NE/350). Written informed consent to participate in this study was provided by the participantsâ legal guardian/next of kin. Written informed consent was obtained from the minor(s)â legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.\n\n\nAuthor contributions\n\nThe study was conceived and designed by JS. JS, LS, EB, TL and GT contributed to the acquisition, analysis of data, and writing the first draft. LS contributed to collecting the data and communicated with the patientâs family. JS contributed to molecular genetic studies and in silico analysis. The final version was edited by JS. All authors edited and approved the final manuscript.",
"appendix": "Acknowledgements\n\nThe authors thank the patients and family members who took part in this study.\n\n\nReferences\n\nBachmann-Gagescu R, Dempsey JC, Bulgheroni S, et al.: Healthcare recommendations for Joubert syndrome. Am J Med Genet A. 2020; 182: 229â249. PubMed Abstract | Publisher Full Text\n\nBachmann-Gagescu R, Dempsey JC, Phelps IG, et al.: Joubert syndrome: a model for untangling recessive disorders with extreme genetic heterogeneity. J Med Genet. 2015; 52: 514â522. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrancati F, Dallapiccola B, Valente EM: Joubert Syndrome and related disorders. Orphanet J Rare Dis. 2010; 5: 20. PubMed Abstract | Publisher Full Text\n\nCarss KJ, Arno G, Erwood M, et al.: Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease. Am J Hum Genet. 2017; 100: 75â90. PubMed Abstract | Publisher Full Text\n\nCideciyan AV, Rachel RA, Aleman TS, et al.: Cone photoreceptors are the main targets for gene therapy of NPHP5 (IQCB1) or NPHP6 (CEP290) blindness: generation of an all-cone Nphp6 hypomorph mouse that mimics the human retinal ciliopathy. Hum Mol Genet. 2011; 20: 1411â1423. PubMed Abstract | Publisher Full Text\n\nCoene KL, Roepman R, Doherty D, et al.: OFD1 is mutated in X-linked Joubert syndrome and interacts with LCA5-encoded lebercilin. Am J Hum Genet. 2009; 85: 465â481. PubMed Abstract | Publisher Full Text\n\nCoppieters F, Casteels I, Meire F, et al.: Genetic screening of LCA in Belgium: predominance of CEP290 and identification of potential modifier alleles in AHI1 of CEP290-related phenotypes. Hum Mutat. 2010; 31: E1709âE1766. PubMed Abstract | Publisher Full Text\n\nFeldhaus B, Weisschuh N, Nasser F, et al.: CEP290 Mutation Spectrum and Delineation of the Associated Phenotype in a Large German Cohort: A Monocentric Study. Am J Ophthalmol. 2020; 211: 142â150. PubMed Abstract | Publisher Full Text\n\nFleming LR, Doherty DA, Parisi MA, et al.: Prospective Evaluation of Kidney Disease in Joubert Syndrome. Clin J Am Soc Nephrol. 2017; 12: 1962â1973. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFokkema IF, Taschner PE, Schaafsma GC, et al.: LOVD v.2.0: the next generation in gene variant databases. Hum Mutat. 2011 May; 32(5): 557â563. PubMed Abstract | Publisher Full Text\n\nFrank V, den Hollander AI , BrÃŒchle NO, et al.: Mutations of the CEP290 gene encoding a centrosomal protein cause Meckel-Gruber syndrome. Hum Mutat. 2008; 29: 45â52. PubMed Abstract | Publisher Full Text\n\nJoubert M, Eisenring JJ, Robb JP, et al.: Familial agenesis of the cerebellar vermis. A syndrome of episodic hyperpnea, abnormal eye movements, ataxia, and retardation. Neurology. 1969; 19: 813â825. PubMed Abstract | Publisher Full Text\n\nKroes HY, Monroe GR, van der Zwaag B , et al.: Joubert syndrome: genotyping a Northern European patient cohort. Eur J Hum Genet. 2016; 24: 214â220. PubMed Abstract | Publisher Full Text\n\nKumar J, Kumar AThe molar tooth sign of Joubert syndrome.Arch Neurol.2007 Apr;64(4):602â603.Publisher Full Text PubMed Abstract |\n\nParisi M, Glass I: Joubert Syndrome. Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews(®). Seattle: University of Washington; 1993.\n\nCopyright ©: University of Washington, Seattle. GeneReviews is a registered trademark of the University of Washington, Seattle. All rights reserved., Seattle (WA).1993-2022.\n\nParisi MA: Clinical and molecular features of Joubert syndrome and related disorders. Am J Med Genet C Semin Med Genet. 2009; 151c: 326â340. PubMed Abstract | Publisher Full Text\n\nPoretti Aet al.: Joubert syndrome and related disorders: spectrum of neuroimaging findings in 75 patients. AJNR Am J Neuroradiol. 2011 Sep;32(8):1459â1463. Publisher Full Text PubMed Abstract | PubMed Abstract |\n\nRomani M, Micalizzi A, Valente EM: Joubert syndrome: congenital cerebellar ataxia with the molar tooth. Lancet Neurol. 2013; 12: 894â905. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSayer JA, Otto EA, O'Toole JF, et al.: The centrosomal protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor ATF4. Nat Genet. 2006; 38: 674â681. PubMed Abstract | Publisher Full Text\n\nSuzuki T, Miyake N, Tsurusaki Y, et al.: Molecular genetic analysis of 30 families with Joubert syndrome. Clin Genet. 2016; 90: 526â535. PubMed Abstract | Publisher Full Text\n\nTallila J, Salonen R, Kohlschmidt N, et al.: Mutation spectrum of Meckel syndrome genes: one group of syndromes or several distinct groups?. Hum Mutat. 2009; 30: E813âE830. PubMed Abstract | Publisher Full Text\n\nTravaglini L, Brancati F, Attie-Bitach T, et al.: Expanding CEP290 mutational spectrum in ciliopathies. Am J Med Genet A. 2009; 149a: 2173â2180. PubMed Abstract | Publisher Full Text\n\nValente EM, Brancati F, Boltshauser E, et al.: Clinical utility gene card for: Joubert syndrome--update 2013. Eur J Hum Genet. 2013; 21. PubMed Abstract | Publisher Full Text\n\nVilboux T, Doherty DA, Glass IA, et al.: Molecular genetic findings and clinical correlations in 100 patients with Joubert syndrome and related disorders prospectively evaluated at a single center. Genet Med. 2017; 19: 875â882. Publisher Full Text"
}
|
[
{
"id": "140267",
"date": "02 Aug 2022",
"name": "Muhammad Ansar",
"expertise": [
"Reviewer Expertise Human Genetics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report, Spahiu et al. presented a patient with a known mutation in CEP290 gene. Mutations in CEP290 are known to cause Joubert syndrome (JS), Meckel syndrome and Leber congenital amaurosis (LCA). Authors concluded that CEP290 variant is recurrent based on its report in two families sharing common ancestry. The report is well written but discussion can be further improved as specified below;\nAuthors should compare the clinical profile of patients from both families carrying c.5493delA.\n\nIt would be nice to add fundus image of this patient.\n\nIt would be better to compare clinical presentation of patients carrying c.5493delA. variant with other published cases carrying loss of function variants in CEP290 gene.\n\nIs the background of the caseâs history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": [
{
"c_id": "9465",
"date": "04 Apr 2023",
"name": "John Sayer",
"role": "Author Response",
"response": "In this case report, Spahiu et al. presented a patient with a known mutation in CEP290 gene. Mutations in CEP290 are known to cause Joubert syndrome (JS), Meckel syndrome and Leber congenital amaurosis (LCA). Authors concluded that CEP290 variant is recurrent based on its report in two families sharing common ancestry. The report is well written but discussion can be further improved as specified below; Authors should compare the clinical profile of patients from both families carrying c.5493delA. Thank you we now provide the clinical profile in a new table 1 of our case with homozygous c.5493delA variant and have added details of our case and additional details for the Kosovar-Albanian cases of MKS to table 2 to allow a direct comparison of features.  It would be nice to add fundus image of this patient. Unfortunately no fundus image is available  It would be better to compare clinical presentation of patients carrying c.5493delA. variant with other published cases carrying loss of function variants in CEP290 gene. Thank you, this is a helpful suggestion. We have added some more detailed discussion comparing our case with other cases with loss of function variants in CEP290 gene. There is no real genotype-phenotype with variants in this gene, even with LoF alleles."
}
]
},
{
"id": "156994",
"date": "14 Dec 2022",
"name": "Karen I. Lange",
"expertise": [
"Reviewer Expertise Genetics/Ciliopathies"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case study describes a two-year-old girl with clinical phenotypes that affect her eyes, kidneys, and brain. The âmolar tooth signâ was observed on MRI and she was diagnosed with Joubert Syndrome. Exome sequencing revealed she has a homozygous mutation in CEP290(c.5493delA/p.A1832fs*19) that she inherited from both parents. This variant has previously been identified in 2 other families from the Kosovo-Albanian region suggesting it may be a recurrent allele in this population. The clinical description of the patient is thorough and the observation that this may be a founder allele in this population is important.\n\nI appreciate that the authors include a table which highlights the spectrum of clinical phenotypes associated with this specific CEP290 variant. It is intriguing that previous reports of individuals that were homozygous for this allele were diagnosed with perinatal lethal Meckel Syndrome while the homozygous patient in this case study is has Joubert Syndrome. It would be worthwhile to add a sentence in the discussion to speculate on why this might be.\nI have some minor recommendations that would help with clarity:\nâBlood from the patient and his healthy parentsâ should be her/their.\n\nâno clear signs of foramina magnum obstruction and a typical âmolar tooth signâ This should be reworded because I interpreted this as no MTS was observed (even though it is clearly shown in Figure 1C)\n\nIn Table 1 It would be more clear if the Clinical Phenotype of COR001 was listed as Joubert Syndrome since COR is a subset of Joubert.\n\nI believe the clinical phenotype of G001347 in Table 1 should be retinal dystrophy not LCA.\n\nIs the background of the caseâs history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": [
{
"c_id": "9466",
"date": "04 Apr 2023",
"name": "John Sayer",
"role": "Author Response",
"response": "I appreciate that the authors include a table which highlights the spectrum of clinical phenotypes associated with this specific CEP290 variant. It is intriguing that previous reports of individuals that were homozygous for this allele were diagnosed with perinatal lethal Meckel Syndrome while the homozygous patient in this case study is has Joubert Syndrome. It would be worthwhile to add a sentence in the discussion to speculate on why this might be. Thank you. We have added a section on genotype phenotype correlation in CEP290. I have some minor recommendations that would help with clarity: âBlood from the patient and his healthy parentsâ should be her/their. Corrected  âno clear signs of foramina magnum obstruction and a typical âmolar tooth signâ This should be reworded because I interpreted this as no MTS was observed (even though it is clearly shown in Figure 1C)  Reworded  In Table 1 It would be more clear if the Clinical Phenotype of COR001 was listed as Joubert Syndrome since COR is a subset of Joubert. Corrected  I believe the clinical phenotype of G001347 in Table 1 should be retinal dystrophy not LCA. Corrected"
}
]
}
] | 1
|
https://f1000research.com/articles/11-388
|
https://f1000research.com/articles/11-1107/v1
|
27 Sep 22
|
{
"type": "Research Article",
"title": "Prevalence of common mental health problems and associated factors among university students visiting Supara mental health service: A cross-sectional study",
"authors": [
"Pantri Kirdchok",
"Varuna Kolkijkovin",
"Wanida Munsukpol",
"Chotiman Chinvararak",
"Pantri Kirdchok",
"Varuna Kolkijkovin",
"Wanida Munsukpol"
],
"abstract": "Background: Early studies found that the mental health problems rate was relatively high in university students. We aimed to investigate the prevalence of mental problems and associated factors in university students. Methods: We conducted a cross-sectional descriptive study at Supara mental health service in the Faculty of Medicine Vajira Hospital between February 2020 to June 2021. The primary outcome was the prevalence of psychiatric diagnosis according to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10). The secondary assessments included the Patient Health Questionnaire-9 (PHQ-9), 8 items from the Mini International Neuropsychiatric Interview (MINI) to assess suicidal risk (8Q), and the Thai Mental Health Indicator (TMHI-15). The prevalence of mental health problems was presented by frequency and percentage. In addition, multivariable regression analysis was used to identify potential predictors of mental health problems.\n\nResults: A total of 184 participants (62% female; mean age = 22.49 years (SD 3.93) were recruited. The depressive disorders, adjustment disorders, and anxiety disorders rates were 57.1%, 15.2% and 13.6%, respectively. Grade point averages (GPAs) below 3.0 (OR=3.09, 95%CI: 1.17-8.14) and a family history of mental disorder (OR=3.40, 95%CI: 1.10-10.48) were significant associated factors of moderate to severe mental health problems. Detecting and screening these factors may help the university to provide early detection and treatment for students. Conclusions: Depressive disorders were the most common mental health disorders. Females, low GPAs and a family history of mental disorder were predictors of moderate to severe mental health problems.",
"keywords": [
"Mental health problems",
"University students",
"Mental health service",
"Thailand"
],
"content": "Introduction\n\nMental health issues are one of the leading global problems among university students [Macaskill, 2013; Akhtar et al., 2020]. Early studies found that mental health problems, especially in medical and nursing students [MacLean et al., 2016, Tung et al., 2018], disturbed not only quality of life but also affected academic performance [Bakker et al., 2020] and empathic feelings for patients [Lins et al., 2015]. In addition, examples of common mental health problems in university students are stress [Pacheco et al., 2017], burnout [Pacheco et al., 2008], sleep problems [Azad et al., 2015; Chinvararak et al., 2021], depression [Tung et al., 2018, Pacheco et al., 2017], anxiety [MacLean et al., 2016; Pacheco et al., 2017], and suicidal ideation [Pacheco et al., 2008]. Prior studies showed that the prevalence of depression in medical students globally and in Thailand ranged between 2-30% [Rotenstein et al., 2016; Phomprasith et al., 2022], while the anxiety rate was approximately 6-78% [Puthran et al., 2016]. Other psychiatric disorders, such as obsessive-compulsive disorder (OCD) and substance use disorder, were also found [Pacheco et al., 2017]. However, most of the results came from a community-based study. There is still limited data on mental health problems in a clinical-based study in which clients decided to consult mental health services.\n\nFurthermore, factors found to be associated with mental health problems in medical students include course year, academic pressure, relationship problems, bullying, sleep deficiency, high workload, and chronic medical diseases [Puthran et al., 2016; Firth, 1986]. However, there are several reports on the prevalence of mental issues; the results revealed that most medical students are more likely not to seek mental health help [Quince et al., 2012]. This cause may result from a negative attitude toward psychiatric disorders, fear of stigmatisation, or confidentiality concern about the treatment process [Jacob et al., 2020; Hankir et al., 2014].\n\nâSuparaâ mental health clinic is a specialised in-house clinic that provides mental health service for students and staff of Navamindradhiraj University. This clinic was established and implemented in 2020 by the department of psychiatry, Faculty of Medicine Vajira Hospital, which is affiliated with Navamindradhiraj University. The clinic intended to increase access to mental health services for university students by addressing confidentiality, privacy, and waiting time. There are psychologists, mental health nurses, and psychiatrists who provide comprehensive mental health evaluation and treatment (Figure 1).\n\nAlthough much research has studied the prevalence and associated factors of mental health problems in students, Supara mental health clinic is a specific service model. Consequently, this study aimed to investigate the prevalence of common mental health problems among undergraduate and postgraduate students of Navamindradhiraj University who decide to seek the mental health service. The results of this study will be used to plan for further service improvements based on the prevalence of common problems and related factors.\n\n\nMethods\n\nWe obtained approval from the Ethical Committee of the Institutional Review Board of the Faculty of Medicine Vajira Hospital on June 26th, 2021 (COA no. 122/64E). All participants were asked to provide written informed consent to complete routine patient-reported outcome measures (PROMs) when using the clinicâs service. Informed consent described the purpose of data collection, i.e. to assess treatment effectiveness in the routine clinic. They were also informed that their information might be used in the future under the supervision of the ethical committee. The data used in this study was retrospectively retrieved from the clinicâs records; participants were not directly involved.\n\nWe conducted a cross-sectional descriptive study based on STROBE guidelines [von Elm et al., 2008]. We retrospectively investigated the mental health record form from Supara mental health service's database. The sample size was calculated according to the Cochrane formula [Wayne, 1995]. The number of students visiting (N) Supara mental health service between February 2020 to June 2021 was 260. The sample size was estimated by p = 0.196 according to the study by Limsricharoen et al. [2014]. Using alpha at 0.05 and error (d) at 0.05, the required sample size was 140.\n\nParticipants were included if they: (a) were a student of Navamindradhiraj university; (b) were aged 18 years and older; (c) had a record of using the mental health service. Participants were excluded if their record data was not sufficient to be analysed.\n\nParticipants visiting Supara mental health service would perform the questionnaires in-person on the first visit. We explored the pre-existing database and created the data record form. The data record form comprised five sections: 1) demographic data including sex, age, admission route to the university, course year, faculty, department, underlying disease, waiting time before receiving the first evaluation, satisfactory scores; 2) clinical characteristics including diagnosis recorded according to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) [World Health Organization, 1992], from a consultant psychiatrist, number of visits to the mental health service, a history of psychiatric treatment and, prescription of psychotropic medications; 3) Clinical Global Impression Scale (CGI); 4) Patient Health Questionnaire-9 (PHQ-9); 5) 8 items from the Mini International Neuropsychiatric Interview (MINI) to assess suicidal risk (8Q); and 6) Thai Mental Health Indicator (TMHI-15).\n\nCGI was used to assess the severity of the illness. It is divided into 7 levels of severity, sorted into ascending severity scores: 1) normal or not at all ill, 2) borderline mentally ill, 3) mildly ill, 4) moderately ill, 5) markedly ill, 6) severely ill, and 7) among the most extremely ill patients [Busner and Targum, 2007].\n\nPHQ-9 Thai version consists of a total of 9 questions. The total score of PHQ-9 is categorised into 4 levels of severity of depression: 1) normal (0â6), 2) mild (7â12), 3) moderate (13â18), and 4) severe (â¥19). The sensitivity and specificity of PHQ-9 to distinguish depression are 84% and 77%, respectively [Lotrakul et al., 2008].\n\n8Q was developed to measure suicidal risk by Kittirattanapaiboon following the MINI. It is classified as suicidal risk into 1) low risk (1-8), 2) moderate risk (9-16), and 3) high risk (>17) [Kittirattanapaiboon and Khamwongpin, 2005].\n\nTMHI-15 short version was developed by Monhkol et al. and aimed to measure the mental health of Thai people. The total scores can be divided into 3 groups: 1) better than average mental health (51-60), 2) average mental health (44-50), and 3) below average mental health (<43) [Mongkol et al., 2013].\n\nAll questionnaires except CGI in this study were self-rated by participants on their first visit to the Supara mental health service. The CGI scale was rated by psychiatrists on the patientâs first visit. PK and CC extracted the record data independently. If there were any conflicts in the data, the third author would double-check the result and provide the final consensus.\n\nData were analysed using SPSS software (version 28.0; IBM, Chicago, IL, USA). The prevalence of mental health problems was presented by frequency and percentage. In addition, associated factors to the severity of mental health problems were analysed by the Independent samples t-test, Mann-Whitney U test, Chi-square test or Fisher's exact test, depending on the variable types. Finally, multivariable regression analysis (odds ratio [OR] and 95% confidence interval [CI]) was used to identify potential predictors of mental health problems. We classified the severity of mental health problems by CGI scale: normal to mild (CGI 1-3) and moderate to severe (CGI 4-7). We used the listwise deletion method for handling missing data. P < 0.05 was considered statistically significant.\n\n\nResults\n\nOf 184 participants recruited in this study, they had a mean age of 22.49 years old (SD 3.92). Most participants were female (62%), undergraduate (84.8%), and studied in the Doctor of Medicine programme. Most medical students were enrolled at the university through the consortium of Thai Medical Schools admission system (71.4%), and they were preclinical year students (59%). Furthermore, the participants' average grade point average (GPAs) was 3.15 (SD 0.42). The median waiting time for their first psychiatric assessment was 0 days (IQR 0-5). Lastly, the majority of participants were satisfied with the university's mental health service, with a mean score of 9.45 (SD 0.83) (Table 1).\n\nTable 2 demonstrates the prevalence of psychiatric diagnoses. Among all psychiatric disorders, depressive disorders (57.1%), adjustment disorders (15.2%), and anxiety disorders (13.6%) were the top three most common diagnoses. The median number of visits to the mental health service was 7 (IRQ 3-14). Approximately 86% of participants received psychotropic medications.\n\nTable 3 reveals the measurement tools used in the mental health service. Around 38% of participants had a CGI scale of 4 (moderately ill). Most participants had mild depression (50%) and low suicidal risk (76.3%) from PHQ-9 and 8Q, respectively. Unsurprisingly, all participants had below-average mental health from TMHI-15.\n\nThe results of multivariable regression analysis showed that GPAs below 3.0 (OR = 3.09, 95%CI: 1.17-8.14) and a family history of mental disorder (OR = 3.40, 95%CI: 1.10-10.48) were the independent predictors of moderate to severe mental health problems (Tables 4 and 5).\n\n1 Crude Odds Ratio estimated by Binary Logistic regression.\n\n2 Adjusted Odds Ratio estimated by Multiple Logistic regression.\n\n\nDiscussion\n\nMental health problems are a common issue in university students. The total number of Navamindradhiraj university students is approximately 2,500. In addition, around 260 students visited Suprara mental health clinic, so the point prevalence of mental health problems is around 10%. Moreover, this number is only for students who sought mental health treatment. Hence, the actual prevalence in a community-based setting is likely to be over 10%. The results revealed that medical students and residents of fellowship are the majority of participants in this study. We believe this is because students in the Faculty of Medicine, students are more familiar with the health service and have more exposure to the psychiatric unit due to their clinical coursework.\n\nThe most common psychiatric diagnoses in our study were depressive disorders, adjustment disorders and anxiety disorders, respectively. Our study is among the first to present the prevalence of common mental health problems from a university mental health service. The prevalence of depression in the present study was higher than in prior studies [Akhtar et al., 2020; Pacheco et al., 2017; Rotenstein et al., 2016], likely because the participants were recruited from the mental health service. However, the anxiety rate was similar to earlier studies [Phomprasith et al., 2022]. Additionally, the three most common mental health problems presented in the service are similar to those in the community-based study of Thai university students [Chiddaycha and Wainipitapong, 2021]. It can also be implied that university mental health service may benefit more from improving the service for depression and anxiety; for example, through the use of standard psychotherapy (cognitive behavioural therapy, interpersonal therapy) [Hofmann et al., 2012].\n\nThe PHQ-9 and 8Q questionnaires showed most participants had mild depression and a low risk of suicide. However, the overall CGI scale at baseline is four (moderately ill), which is clinically significant and needs to be tackled to prevent further complications and disease progression. All participants had below-average mental health from TMHI-15. However, TMHI-15 could be useful for monitoring treatment improvement in mental health status.\n\nInterestingly, most clients rated our mental health service a very high satisfaction score. In addition, it can be seen that the median waiting time for the first psychiatric evaluation was zero days as the university policy intends to provide a standard of mental health care urgently and vitally for the students. Furthermore, more than half of our participants would like to use telemedicine. This is probably influenced by the COVID-19 pandemic. However, it can indicate that the clinic should implement telemedicine in the service.\n\nThe multivariable regression analysis demonstrated that having a low GPAs and a family history of mental disorders were strong predictors of moderate to severe mental health problems. Therefore, these factors may be helpful in screening mental health problems for university students.\n\nWe are aware of some limitations of this study. First, due to the cross-sectional descriptive design, we can only indicate associated factors, not causal relationships. Secondly, the results of the present came from Navamindradhiraj University, so it may have a different context from other universities, which may not be representative of all university students.\n\nFuture research should investigate the longitudinal data that can assess the effectiveness of our mental health service. Moreover, a study about mental health problems in university staff should be done because Supara mental health service also provides mental health consults to all university staff.\n\n\nConclusions\n\nThe most common mental health problems in university students were depressive disorders. Low GPAs and a family history of mental disorder were independent predictors of moderate to severe mental health problems. Screening these factors may be helpful for the university to provide early detection and management.\n\n\nData availability\n\nfigshare: Data-Supara Mental Health Service. https://doi.org/10.6084/m9.figshare.21131953.v2 (Chinvararak, 2022)\n\nThis project contains the following underlying data:\n\n- Raw data-Pre-analysed.xlsx (Anonymised responses in excel sheet)\n\nfigshare: Data-Supara Mental Health Service. https://doi.org/10.6084/m9.figshare.21131953.v2 (Chinvararak, 2022)\n\nThis project contains the following extended data:\n\n- Data Record Form_Supara.docx (Blank English copy of the data record form used in this study)\n\n- Figure 1. jpg",
"appendix": "Acknowledgements\n\nThe authors acknowledge all study participants and would like to thank Mr Anucha Kamson for his assistance in the statistical analysis.\n\n\nReferences\n\nAkhtar P, Ma L, Waqas A, et al.: Prevalence of depression among university students in low and middle income countries (LMICs): a systematic review and meta-analysis. J. Affect. Disord. 2020; 274: 911â919. PubMed Abstract | Publisher Full Text\n\nAzad MC, Fraser K, Rumana N, et al.: Sleep disturbances among medical students: a global perspective. J. Clin. Sleep. Med. 2015; 11(1): 69â74. PubMed Abstract | Publisher Full Text\n\nBakker E, Kox J, Boot C, et al.: Improving mental health of student and novice nurses to prevent dropout: A systematic review. J. Adv. Nurs. 2020; 76(10): 2494â2509. PubMed Abstract | Publisher Full Text\n\nBusner J, Targum SD: The clinical global impressions scale: applying a research tool in clinical practice. Psychiatry (Edgmont (Pa.: Township)). 2007; 4(7): 28â37. PubMed Abstract\n\nChiddaycha M, Wainipitapong S: Mental health among Thai medical students: Preadmission evaluation and service utilisation. Health Sci. Rep. 2021; 4(4): e416. PubMed Abstract | Publisher Full Text\n\nChinvararak C, Kirdchok P, Pruttithavorn W, et al.: Sleep Quality and Associated Factors in Preclinical Medical Students in Faculty of Medicine Vajira Hospital. Vajira Medical Journal: Journal of Urban Medicine. 2021; 65(4): 332â342.\n\nChinvararak C: Data-Supara Mental Health Service. figshare. [Dataset].2022. Publisher Full Text\n\nFirth J: Levels and sources of stress in medical students. Br. Med. J. (Clin. Res. Ed.). 1986; 292(6529): 1177â1180. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHankir AK, Northall A, Zaman R: Stigma and mental health challenges in medical students. BMJ Case Rep. 2014; 2014: bcr2014205226. 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JAMA. 2016; 316(21): 2214â2236. PubMed Abstract | Publisher Full Text\n\nTung YJ, Lo K, Ho R, et al.: Prevalence of depression among nursing students: A systematic review and meta-analysis. Nurse Educ. Today. 2018; 63: 119â129. PubMed Abstract | Publisher Full Text\n\nvon Elm E , Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. J. Clin. Epidemiol. 2008; 61(4): 344â349. Publisher Full Text\n\nWayne WD: Biostatistics: a foundation for analysis in the health sciences. (6th ed).New York:Wiley & Sons;1995.\n\nWorld Health Organization: The ICD-10 classification of mental and behavioural disorders: clinical descriptions and diagnostic guidelines. World Health Organization;1992.Reference Source"
}
|
[
{
"id": "152313",
"date": "09 Nov 2022",
"name": "Thanapob Bumphenkiatikul",
"expertise": [
"Reviewer Expertise Transgender Healthcare",
"Medical Education"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes common mental health problems and associated factors among university students visiting one in-house mental health clinic that provides services for students and staff of one university in Thailand. The results show depressive disorder, adjustment disorder, and anxiety disorder as the three most common psychiatric diagnoses among participants, which are similar to those in the community-based study in another Thai medical school. The study also highlights the association between lower grade point averages and a family history of mental disorders with moderate to severe mental health problems and suggests using these factors as a screening tool for mental health problems among university students.\n\nThe clinic was established and implemented in February 2020 to serve approximately 2,500 students and an additional number of all university staff. Despite caring for 260 students in 17 months, the waiting time for the first psychiatric evaluation was surprisingly reported as zero days. It would be more informative for readers if the authors mentioned the number of psychologists, mental health nurses, and psychiatrists providing care at the clinic, the total number of university staff and those seeking care at the clinic, the number of visits by several types of clients during the same 17 months period and statistics of the number of diagnoses given to each clients receiving the service. In the follow-up study, with an increasing number of clients, it would be interesting to investigate and compare the results between every subgroup, including undergraduate and postgraduate students, or medical and nursing students.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9008",
"date": "17 Nov 2022",
"name": "Chotiman Chinvararak",
"role": "Author Response",
"response": "Thank you very much for your review and suggestions for our manuscript. In the follow-up study, which will have more participants, we plan to investigate the subgroup and make a comparison between undergraduate and postgraduate, between the faculty and the route of admission. We will design further study with the valuable recommendation from the reviewer. We appreciate your time reviewing, which assists in making the manuscript a better quality publication. Best regards Chotiman Chinvararak"
}
]
},
{
"id": "162187",
"date": "20 Mar 2023",
"name": "Pracheth Raghuveer",
"expertise": [
"Reviewer Expertise Maternal Mental Health",
"Non-Communicable Diseases",
"Geriatrics."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study highlights the prevalence and determinants of mental health problems among university students in a specialized mental health clinic. It is a well-written paper. There is further scope for improvement. Here are my constructive comments:\nThe authors mention that the majority of the participants were satisfied with the mental health services. But it is unclear how satisfaction was assessed. Was there a scale used? Please explain in the methods section.\n\nThe discussion needs major improvement. Please compare your findings with the findings of other studies. Speculate wherever necessary.\n\nThe strengths of this study or the novelty must be explained in the discussion.\n\nMost of the assessments were done using self-rated scales. This must be mentioned as a limitation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9488",
"date": "03 Apr 2023",
"name": "Chotiman Chinvararak",
"role": "Author Response",
"response": "Thank you very much for your time and valuable comment in order to improve our manuscript. We really appreciate it. We have addressed all of your comments in the 2nd version of the manuscript. Sincerely yours Chotiman Chinvararak Coressponding author"
}
]
}
] | 1
|
https://f1000research.com/articles/11-1107
|
https://f1000research.com/articles/12-356/v1
|
31 Mar 23
|
{
"type": "Research Article",
"title": "Success rate of repeated cycles of induction in labour in a UK clinical setting: A cohort study",
"authors": [
"Anas Alojayli",
"Rahim Haloob",
"Rahim Haloob"
],
"abstract": "Background: Births are increasingly started artificially using medications such as prostaglandin, oxytocin, or other mechanical methods, a process known as induction of labour (IOL). The aim of the study was to identify the success rate of a repeat cycle of induction of labour (IOL) with prostaglandins, and any association between smoking and patient response to prostaglandin. Methods: This was a retrospective cohort study set in Basildon and Thurrock University Hospital, UK. Â IOL data from patients were collected between 1 January 2021 and 30 March 2021. Data were retrieved from hospital records and categorised by prostaglandin cycle(s), administration of oxytocin, artificial rupture of membranes (ARM), spontaneous rupture of membranes (SROM), smoking status, patient BMI, age, ethnicity, gestation age, Bishop score, and delivery method (assisted, unassisted, or Caesarean (C-)section). The data were analysed using Chi-square, binomial and multinomial logistic regression. The success rate was interpreted from relative frequencies of delivery method (unassisted, assisted or C-section). Results: Unassisted vaginal delivery (n =121; 48.0%) was the most common outcome with prostaglandin IOL followed by C-section (n = 105 41.7%). Only 10.3% had an assisted vaginal delivery. Of those who had a repeated IOL cycle, 85.7% had a C-section. There was no difference in prostaglandin administration by smoking status or any association between smoking status and mode of delivery in IOL. Conclusions: Repeat cycle of IOL does not enhance the vaginal delivery with only 14.3% unassisted vaginal births. There was no evidence that smoking impacted on response to prostaglandins or method of delivery.",
"keywords": [
"artificial rupture of membranes",
"C-section",
"repeat induction of labour",
"spontaneous rupture of membranes",
"unassisted (normal)",
"assisted vaginal delivery (instrumental delivery)."
],
"content": "Introduction\n\nBirths are increasingly started artificially using medications such as prostaglandin, oxytocin, or other mechanical methods. This process is known as induction of labour (IOL). IOL typically involves a combination of medications known as dinoprostones that are inserted into the vagina, causing artificial rupture of the membranes (âreleasing the watersâ), and administration (via a drip) of the synthetic hormone oxytocin.\n\nThis retrospective cohort study focused on the success rate of IOL with a repeated cycle of cervical ripening with dinoprostone prostaglandin E2 as vaginal pessary 10mg (Propess) and dinoprostone prostaglandin E2 as vaginal tablet 3mg (Prostin E2).\n\nFurthermore, the study looked for any associations between smoking, maternal characteristics, and cervical Bishop score and success rate of repeated cycles of IOL.\n\nIOL aims to achieve cervical ripening and progressive uterine contractions leading to progressive dilatation of the cervix to facilitate childbirth. In the UK, between 2007 and 2017, IOL interventions increased by nearly 10% to a frequency of 25.5%. This increase has been driven by the increasing prevalence of medical complications during pregnancy leading to elective IOL prior to 42 weeks of gestation.1\n\nStudies which investigated repeated cycle of induction have been inconsistent in finding a common ground and defining clear guidelines for how to reduce failure of IOL. NICE guidelines2 recommend that if IOL fails, a decision about further management should be made in accordance with the motherâs wishes and clinical circumstances: NICE2 recommends offering a rest period, a further attempt to induce, or a C-section.\n\nSuccessful IOL largely depends on the status of the cervix prior to onset of labour (Bishop score). Inducing labour when the Bishop score is low will lead to an increased rate of C-section. The incidence of adverse outcomes following IOL is highest in nulliparous women who have an unfavourable cervix. For a woman with an unfavourable cervix, IOL interventions such as oxytocin administration or artificial ruptured of membrane (ARM) are associated with reduced effectiveness and high failure rates. Prostaglandin medications are frequently used in the process of IOL. Understanding the history and research that supports prostaglandin use for IOL is crucial for safe practice.3 We attempted to look at IOL interventions to find out if there is an association between administration of prostaglandin for IOL and mode of delivery.\n\nSmoking may influence metabolism of prostaglandin and consequently the response to prostaglandin during IOL.4,5 The increased risk of preterm labor and delivery in smokers is related to increased contractile sensitivity and activity of the uterine myometrium4 upon exposure to PG F2α through the respective receptors. Although smoking during pregnancy is a major risk factor for preterm delivery, the underlying mechanism by which smoking stimulates uterine contractions is still poorly understood. In one study, Nakamoto et al.4 showed that cigarette smoke extract enhances oxytocin-induced rhythmic contractions of rat and human preterm myometrium. In this study, we explored whether smoking status was associated with outcome of IOL with prostaglandins.\n\nThis study aimed first to determine whether mode of delivery (normal vaginal delivery versus C-section and assisted vaginal delivery) varied between labours with and without a repeated cycle of IOL, with 10 mg dinoprostone prostaglandin E2 vaginal pessary and 3 mg dinoprostone prostaglandin E2 vaginal tablet. Secondly, potential associations between smoking and response to prostaglandin were explored. Further, any associations between motherâs age, ethnicity, BMI, Bishop score, gestational age and IOL outcome were explored, as well as between IOL intervention and delivery method. Finally, the data were analysed to identify the most common reason for IOL.\n\n\nMethods\n\nThis retrospective cohort study was approved by the audit team at Basildon University Hospital on 23 September 2021. The study was a retrospective audit proved by audit committee in obstetrics and Gynaecology department at Basildon University Hospital and the identity of the patients was not revealed, therefore, there was no need for consent.\n\nDinoprostone (Prostaglandin E2 Propess 10 mg vaginal delivery system: Ferring Controlled Therapeutics Ltd, or Prostin E2 3 mg vaginal delivery tablets: Pfizer) were the standard care for cervical ripening in full-term pregnancies in this cohort study.\n\nData were collected from electronic medical records and notes. Data from women who had experienced IOL at Basildon University Hospital, UK, between 1 January 2021 and 30 March 2021 were included in the analysis. Data for IOL which resulted in stillbirth or in the birth of twins were not included. This retrospective cohort study was approved by the audit team at Basildon University Hospital on 23 September 2021.\n\nSuccess rate in this study is described by the mode of delivery (unassisted vaginal births or C-section). We researched mode of delivery with and without a repeat cycle of IOL. Data were categorised for exploration by: ARM, spontaneous rupture of membrane (SROM), prostaglandin and/or oxytocin administration, and smoking status. Demographic data collected and analysed for associations with the outcomes included motherâs age, ethnicity, BMI, Bishop score, and gestational age. Finally, data on reasons for IOL were collated.\n\nThe data were imported into SPSS. Thereafter The data were analysed statistically using Chi-square, binomial, and multinomial logistic regression.\n\nNumeric variables were created by separating out methods. Chi-square test and multinomial logistic regression with delivery type (three (3) categories).\n\nWe used a binomial one-sample test comparing each sample delivery method to the related population rate. Moreover, A Chi-square test was carried out to compare rates for all three sample delivery methods to the population rates at the same time.\n\nThe most common reasons for IOL are presented in Table 2.\n\n\nResults\n\nOf the 252 women in the sample, 86.5% received at least one form of prostaglandin (n = 218; see Table 1). There was close to an even split of ARM (n = 137; 54.4%) versus SROM. The most common type was unassisted vaginal delivery (n = 121; 48.0%), followed by C-section (n = 105; 41.7%), while only 10.3% had an assisted vaginal delivery (n = 10.3%). A little over half the sample had at least one partner who had smoked at some point (n = 129; 51.2%). The sample population was predominantly White (n = 209; 82.9%). Reasons for IOL are presented in Table 2. Means and standard deviations are reported for motherâs age, BMI, Bishop score, and gestational age in Table 3.\n\nARM: artificial rupture of membranes; SROM: spontaneous rupture of membranes.\n\nLGA: large for gestational age; Raised UPCR: raised urine protein creatinine ratio.\n\nSD: standard deviation; SE: Standard error; BMI: body mass index.\n\nA chi-square test was completed to explore associations between repeated IOL with prostaglandin and delivery outcome. There was a significant difference in delivery method between patients with repeated IOL cycles and those with either one cycle of prostaglandins or no prostaglandins (Ï2(2) = 5.802, p = 0.055). Of those who had a repeated IOL cycle, 85.7% had a C-section (see Table 4). This was marginally significant, likely because of the small sample size (n = 7).\n\nRepeated: repeated cycle of induction; C-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery).\n\nChi-square tests showed no difference in delivery mode between one cycle of prostaglandin compared to none (Ï2(2) = 1.367, p = 0.505; see Table 5), or between ARM and SROM (Ï2(2) = 3.038, p = 0.219; see Table 6). Further, for those who had received at least one type of prostaglandin, there was no difference in delivery method between ARM and SROM (Ï2(2) = 2.651, p = 0.266; see Table 7).\n\nC-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery); C-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery).\n\nARM: artificial rupture of membranes; PROM: prelabour rupture of membranes.\n\nChi-square tests showed a significant difference in delivery mode following administration of prostaglandin, with and without administration of oxytocin (Ï2(2) = 11.221, p = 0.004). There was a higher number of unassisted vaginal deliveries without oxytocin administration compared to those who had received oxytocin (see Table 8).\n\nC-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery).\n\nHowever, for those who had received at least one type of prostaglandin and who had experienced SROM, there was no significant difference in delivery mode between cases with or without oxytocin (Ï2(2) = 1.241, p = 0.538; see Table 9). Similarly, for those who had experienced SROM and no type of prostaglandin, there was no significant difference in delivery mode with or without oxytocin (Ï2(2) = 0.699, p = 0.705; see Table 10).\n\nC-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery).\n\nC-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery).\n\nThere was no difference in prostaglandin administration by smoking status (Ï2(1) = 0.071, p = 0.790; see Table 11), nor was there any association between smoking status and mode of delivery in IOL with prostaglandins.\n\nProsta: prostaglandins; Smok: smoking.\n\nA multinomial logistic regression was planned for the three delivery modes as the dependent variable, and prostaglandins, smoking status, and their interactions as the independent variables. However, there were insufficient assisted vaginal deliveries, both without prostaglandins treatment and with no smoking background (see Table 12). To conduct the moderation analysis, a binomial logistic regression was therefore completed. This model used two delivery methods (C-section and unassisted vaginal delivery) as the dependent variables, with the same set of independent variables. The Hosmer and Lemeshow goodness-of-fit test was used to assess how well the model fit the data. The test suggested that the model might not be the most appropriate fit for the data (p = 0.999). The model predicted 58.5% of deliveries correctly if all cases had the outcome of unassisted vaginal delivery (see Table 13). The model was not able to increase this classification percentage when adding prostaglandins, smoking, and their interaction as predictors. Collectively, the set of predictors only accounted for 1.2% of the variation in delivery method (Nagelkerke R2 = 0.012). There was no predictive effect of prostaglandins, smoking, or their interaction for delivery mode (see Table 14). Therefore, there was no evidence for a moderating effect of smoking on the relationship between prostaglandins and delivery mode.\n\nC-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery).\n\nC-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery).\n\nCI: confidence interval; df: degrees of freedom; Smok: smoking; Prosta: prostaglandins.\n\nWe conducted a multinomial logistic regression with method of delivery as the dependent variable, and maternal age, Bishop score, BMI, and gestational age as continuous predictors, and ethnicity as a categorical predictor. Unassisted vaginal delivery was used as the reference group. A Pearson Chi-square goodness-of-fit test suggested that the model was a good fit for the data (p = 0.465). Additionally, the model predicted delivery method significantly better than a model containing only the intercept (p = 0.003). Collectively, the set of predictors accounted for 13.7% of the variation in delivery method (Nagelkerke R2 = 0.137). The model was able to correctly classify 53.3% of cases (see Table 15). There was an overall main effect of mothersâ age at delivery (Ï2(2) = 10.672, p = 0.005), Bishop score (Ï2(2) = 11.391, p = 0.003), and gestational age (Ï2(2) = 6.207, p = 0.045; see Table 16). The effect of each predictor was examined by comparing each delivery method to unassisted vaginal delivery (see Table 17). For every year that a motherâs age increased, the odds of delivering by C-section rather than by unassisted vaginal delivery increased by 1.096 (p = 0.002). However, as the Bishop score increased, the likelihood of delivering by C-section decreased compared to unassisted vaginal delivery (p = 0.021). A similar effect was found where assisted vaginal delivery was less likely compared to unassisted vaginal score as the Bishop score increased (p = 0.004). For every one day increase in gestational age, there was an increase in the odds of having an assisted rather than unassisted vaginal delivery (p = 0.030). There were no effects of ethnicity or BMI in predicting mode of delivery.\n\nC-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery).\n\nBMI: body mass index; df: degrees of freedom.\n\nC-sec: Caesarean section; UVag: unassisted vaginal delivery (Normal delivery); AVag: assisted vaginal delivery (instrumental vaginal delivery); Gest age: gestational age; BMI: body mass index.\n\nA binomial one-sample test showed no difference between sample rate of C-sections (0.417; CI: 0.355-0.480) and the population rate of C-sections for the hospital (0.379; p = 0.121).\n\nHowever, the binomial one-sample test showed that there was a marginally lower rate of standard vaginal deliveries in the sample (0.480; CI: 0.417-0.544) compared to the population rate of standard unassisted deliveries for the hospital (0.528; p = 0.072). Further, a binomial one-sample test showed no difference between the sample rate of assisted vaginal deliveries (0.103; CI: 0.069-0.148) and the population rate of assisted deliveries for the hospital (0.092; p = 0.307). Finally, a Chi-square one-sample test showed no difference in the sample compared to the hospital population between rates for all three types of delivery (Ï2(2) = 2.375, p = 0.305; see Figure 1).\n\nOur study showed the C-section rate after repeated cycles of IOL was 87.5%. Moreover, there was no difference in delivery method between ARM and SROM. There was, however, a significant difference in delivery mode for those who received prostaglandins, with or without oxytocin (Ï2(2) = 11.221, p = 0.004), with a higher number of unassisted vaginal deliveries for those who had not received oxytocin (see Table 8).\n\nWith SROM, there was no significant difference between mode of delivery with or without oxytocin.\n\nThere was no evidence of the influence of smoking on the use of prostaglandins and mode of delivery.\n\nRegarding demographic factors, neither ethnicity nor BMI predicted mode of delivery. Every year of maternal age, however, increased the odds of delivering by C-section. Further, every one day increase in gestational age increased the odds of having an assisted compared to unassisted vaginal delivery (p = 0.030). Assisted vaginal delivery was less likely than unassisted vaginal delivery as the Bishop score increased (p = 0.004).\n\nThe most common causes for IOL in this study sample were diabetes, gestational diabetes mellitus, postdates, reduced foetal movements, and prolonged ARM.\n\n\nDiscussion\n\nThis study addressed the value of repeating an IOL cycle, studying the delivery outcomes which may have economic factors related to costs of extended hospital stays. Apart from any influence on the delivery outcome, there may also be an emotional and physical impact of repeating IOL cycles on the mother. We found that a repeated IOL cycle does not enhance the unassisted vaginal delivery rate (with only 14.3% achieving an unassisted vaginal birth). Moreover, for patients over 40 years old treated with repeated IOL cycles, most cases resulted in C-sections.\n\nOther methods of IOL studied included ARM and augmentation with oxytocin; associations between these treatments, and also SROM and mode of delivery were explored. For patients who experienced SROM, there was no significant difference in delivery method with or without oxytocin treatment. Further, there was no difference in delivery mode for those who experienced ARM or SROM.\n\nHowever, there was a significant difference in delivery method with or without oxytocin, with more unassisted vaginal deliveries for those who had not received oxytocin compared to those who had received oxytocin (see Table 8).\n\nThere was no association between smoking, prostaglandin use, and mode of delivery. Smoking does not seem to increase the failure rate of cervical ripening with prostaglandin.\n\nThere was an overall main effect of motherâs age at delivery and gestational age (see Table 16). The effect of each predictor was examined by comparing each delivery method to unassisted vaginal delivery (see Table 17). For every year that a motherâs age increased, the odds of delivering by C-section compared to unassisted vaginal delivery increased by 1.096 (p = 0.002). However, as the Bishop score increased, the likelihood of delivering by C-section decreased compared to unassisted vaginal delivery (p = 0.021). For every one-day increase in gestational age, there was an increase in the odds of having an assisted compared to unassisted vaginal delivery (p = 0.030). Neither ethnicity nor BMI predicted delivery method.\n\nThere was no difference between the rate of C-sections in the sample or hospital population, but a marginally lower rate of standard vaginal deliveries in the sample (0.480; CI: 0.417-0.544) compared to the hospital population rate (0.528; p = 0.072).\n\nFirst, to our knowledge, this is one the first studies to address repeated IOL cycles (Propess + Prostin E2) and success rate in terms of delivery method. Second, we did not find any assosiation between smoking and the effects of prostaglandin in IOL. Third, we explored other potential influences such as ARM, SROM, and oxytocin administration. Lastly, we tested for influences of demographic factors on the success of IOL.\n\nHowever, the research was limited by the small sample size of 252 IOL patients, the small number of repeat cycles of IOL, the short study period and lack of mechanical method of IOL, such as Dilapan-S (an osmotic hygroscopic dilator produced from a patented Aquacryl hydrogel that guarantees consistency of action) and Folly catheter.\n\nRepeated cycles of IOL impact on mothers emotionally, psychologically, and physically, as well as on NHS resources. If IOL fails, NICE guidelines recommend a decision about further management should be made in accordance with the motherâs wishes and clinical circumstances. However, our retrospective study showed that repeated cycles of IOL with prostaglandins failed in most cases and resulted in C-sections. It is possible, however, that alternative mechanical methods (e.g. Dilapan-S® and Foley catheter) offer similar efficacy and better safety compared to PGE2.6 Alternatively, proceed with C-section. Smoking showed no influence on prostaglandin in IOL although further research is needed.\n\n\nConclusions\n\nIn the sample studied, a repeated IOL cycle did not enhance vaginal delivery and further, unassisted (normal) vaginal deliveries were more frequent where prostaglandins had been administered without oxytocin. Smoking was not shown to influence association failed induction of labour with prostaglandins, although a larger study is needed.\n\n\nAuthor contributions\n\nAnas Alojayli (AA) proposed the original idea for the study. AA and Rahim Haboob (RH) designed the retrospective cohort study. AA and RH performed the analyses. AA drafted the article, which was reviewed and revised by both authors.",
"appendix": "Data availability\n\nFigshare: Success rate of repeated cycles of induction in labour in a UK clinical setting: A cohort study, https://doi.org/10.6084/m9.figshare.21534093.v2. 7\n\nThis project contains the following data:\n\n- Anas_Excel.xls\n\n- Anas_RatesOutput.spv\n\n- Anas_SPSSData.sav\n\n- Anas_RatesOutput.spv\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank the midwife, Sarah Leach, for her data collection and her participation. We also acknowledge the input of Dr Claire Yee and thank her for her support with the statistical analysis for this study and Dr. Daniel Rolnik for his review the article, and Nour Alojayli for her data collection and participation.\n\n\nReferences\n\nCarlson N, Ellis J, Page K, et al.: Review of evidence-based methods for successful labor induction. J. Midwifery Womens Health. 2021 Jul; 66(4): 459â469. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNICE: Guideline NG207 Inducing labour.2021.Reference Source\n\nYount SM, Lassiter N: The pharmacology of prostaglandins for induction of labor. J. Midwifery Womens Health. 2013 Mar; 58(2): 133â144. Publisher Full Text\n\nNakamoto T, Yasuda K, Yasuhara M, et al.: Cigarette smoke extract enhances oxytocin-induced rhythmic contractions of rat and human preterm myometrium. Reproduction. 2006 Aug 1; 132(2): 343â353. Publisher Full Text\n\nTakahashi K, Diamond F, Bieniarz J, et al.: Uterine contractility and oxytocin sensitivity in preterm, term, and postterm pregnancy. Am. J. Obstet. Gynecol. 1980; 136: 774â779.\n\nChodankar R, Sood A, Gupta J: An overview of the past, current and future trends for cervical ripening in induction of labour. Obstet. Gynaecol. 2017 Jul; 19(3): 219â226. Publisher Full Text\n\nAlojayli A, Haloob R:Success rate of repeated cycles of induction in labour in a UK clinical setting: A cohort study. [Data set]. figshare. 2022. Publisher Full Text"
}
|
[
{
"id": "173330",
"date": "06 Jul 2023",
"name": "Anna Maria Marconi",
"expertise": [
"Reviewer Expertise Obstetrics",
"Labor and delivery",
"maternal-fetal medicine"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents the results of a retrospective study in which the Authors evaluated the success of repeated prostaglandin E2 applications in women undergoing induction of labor [IOL] and the possible association between cigarette smoking and response to prostaglandins. Despite the high and useless number of tables [there are 17] the Authors studied only 252 women of which 34 did not receive any prostaglandins and therefore it is not clear why they were included in the analysis.\nThere are a number of concerns to address:\n\nTable 2 is an endless list of indications for IOL which, however, are repeated continuously [for example large for gestational age is described as: LGA; large for gestational age; large for dates; fetal problem lga; and so on]; the same happens for many other indications;\n\nThe number of bibliographic references is very small compared to the statements that are made, especially in the introduction, often without any foundation [for example page 3 line 2 from \"IOL typically involves\" ... to \"waters\"]; or in the sixth paragraph where each sentence would need a reference, which does not exist; just to name a few. (Please see Marconi AM, 2019 1).\n\nIt is not clear why all the analyses were carried out, please clarify this. The impact of parity on the results has not been evaluated [perhaps the most important characteristic in women at IOL].\n\nIn summary, I am uncertain why this manuscript is classified as a 'research article'. 252 [minus 34] women were studied, when, as the same Authors state, the induction of labor nowadays involves one out of four women. The majority of the results are well known to the scientific community and, if the references had been chosen carefully, it would be clear that the results being presented have been known for a long time.\n\nFinally, the Authors wanted to study the association between smoking and response to PG, with 13.9% of the data missing; 34.9% of women non-smokers. The fact that there is no association is not surprising, the opposite would have been true.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "209045",
"date": "23 Nov 2023",
"name": "Chanderdeep Sharma",
"expertise": [
"Reviewer Expertise Obstetrics",
"delivery Labor",
"High risk pregnancy"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is poorly conceived. Only three months data was taken. Among 252 women, 34 did not receive any agent for IOL. Large number of tables added unnecessarily. Technically same agent PG E2 used for repeat cycle of IOL. If one method has failed to achieve the desired result, another different method should have been used (preferably mechanical dilatation of cervix with foley's catheter). Parity of women not evaluated (significant factor effecting mode of delivery). They compared effect of smoking, but 14% data is missing. Of all 34 % women are non-smokers, and they have deduced there is no association, which is incorrect.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-356
|
https://f1000research.com/articles/12-355/v1
|
30 Mar 23
|
{
"type": "Data Note",
"title": "The identification of high-preforming antibodies for Ubiquilin-2 for use in Western Blot, immunoprecipitation, and immunofluorescence",
"authors": [
"Ian McDowell",
"Riham Ayoubi",
"Maryam Fotouhi",
"Kathleen Southern",
"Peter S. McPherson",
"Carl Laflamme",
"NeuroSGC/YCharOS/EDDU collaborative group",
"ABIF Consortium",
"Ian McDowell",
"Riham Ayoubi",
"Maryam Fotouhi",
"Kathleen Southern",
"Peter S. McPherson"
],
"abstract": "Ubiquilin-2, a member of the ubiquilin protein family, plays a role in the regulation of various protein degradation pathways, and is mutated in some neurodegenerative diseases. Well-characterized anti-Ubiquilin-2 antibodies would advance reproducible research for Ubiquilin-2 and in turn, benefit the scientific community. In this study, we characterized ten Ubiquilin-2 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.",
"keywords": [
"Uniprot ID Q9UHD9",
"UBQLN2",
"Ubiquilin-2",
"antibody characterization",
"antibody validation",
"Western Blot",
"immunoprecipitation",
"immunofluorescence"
],
"content": "Introduction\n\nUbiquilin-2, a protein encoded by the UBQLN2 gene, plays a critical role in protein degradation pathways; including the ubiquitin-proteasome system (UPS), autophagy and the endoplasmic reticulum-associated protein degradation (ERAD) pathway.1\n\nDisease-causing variants of UBQLN2 have been identified in patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD).2 These UBQLN2 mutations are predicted to be acting on ALS pathological mechanisms by causing UPS and autophagy dysfunction,3 neuroinflammation4,5 and/or formation of stress granules.6,7 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.\n\nHere, we compared the performance of a range of commercially available antibodies for Ubiquilin-2 and validated high-quality antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of Ubiquilin-2 properties and function.\n\n\nResults and discussion\n\nOur standard protocol involved comparing readouts from wild-type (WT) and knockout (KO) cells.8,9 The first step was to identify a cell line(s) that expresses sufficient levels of Ubiquilin-2 to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express UBQLN2 at levels greater than 2.5 log2 (transcripts per million âTPMâ +1), which we had found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HAP1 cells expressed the UBQLN2 transcript at RNA levels above the average range of cancer cells analyzed. The parental and UBQLN2 KO HAP1 cells were obtained from Horizon Discovery (Table 1).\n\nFor Western Blot experiments, we resolved proteins from WT and UBQLN2 KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).\n\nLysates of HAP1 wild-type (WT) and UBQLN2 knockout (KO) were prepared, and 30 ÎŒg of protein were processed for Western Blot with the indicated Ubiquilin-2 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. When the concentration was not indicated by the supplier, which was the case for antibody 35-4400*, we tested it at 1/1000. Antibody dilution used: ab190283* at 1/2000, A9568 at 1/1000, ARP59353 at 1/500, NBP2-25164* at 1/2000, 85509** at 1/1000, PCRP-UBQLN2-1C7* at 1/26, 23449-1-AP at 1/1000, Z-UBQLN2-7** at 1/653, 35-4400* at 1/1000, and 37-7700* at 1/2000. Predicted band size: 65 kDa. *Monoclonal antibody, **Recombinant antibody.\n\nFor immunoprecipitation experiments, we used each of the antibodies to immunopurify Ubiquilin-2 from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the Ubiquilin-2 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).\n\nHAP1 lysates were prepared, and IP was performed using 2.0 ÎŒg of the indicated Ubiquilin-2 antibodies pre-coupled to Dynabeads protein G or protein A or Flag-M2 magnetic beads. Samples were washed and processed for Western Blot with the indicated Ubiquilin-2 antibody. For Western Blot, 85509** was used at 1/1000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.\n\nFor immunofluorescence experiments, as described previously, antibodies were screened using a mosaic strategy.10 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis biases (Figure 3).\n\nHAP1 wild-type (WT) and UBQLN2 knockout (KO) cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with glass bottom. Cells were stained with the indicated Ubiquilin-2 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When the concentration was not indicated by the supplier, which was the case for antibodies ARP59353, 85509**, PCRP-UBQLN2-1C7* and Z-UBQLN2-7**, we tested them at 1/500, 1/200, 1/20 and 1/600, respectively. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab190283* at 1/1000, A9568 at 1/2000, ARP59353 at 1/500, NBP2-25164* at 1/1000, 85509** at 1/200, PCRP-UBQLN2-1C7* at 1/20, 23449-1-AP at 1/300, Z-UBQLN2-7** at 1/600, 35-4400* at 1/500, and 37-7700* at 1/500. Bars = 10 ÎŒm. *Monoclonal antibody, **Recombinant antibody.\n\nIn conclusion, we have screened Ubiquilin-2 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-performing antibodies under our standardized experimental conditions. The underlying data is uploaded to Zenodo.16,17\n\n\nMethods\n\nAll Ubiquilin-2 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure the antibodies are cited properly.11 Peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies are from Thermo Fisher Scientific (cat. number 62-6520 and 65-6120). Peroxidase-conjugated monoclonal anti-Flag M2 is from MilliporeSigma (cat. number A8592). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429). The anti-FLAG (M2 clone) conjugated with Cy3 is from MilliporeSigma (cat. number A9594).\n\n* Monoclonal antibody.\n\n** Recombinant antibody.\n\nBoth HAP1 WT and UBQLN2 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.12 Cells were cultured in DMEM high glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065), 100 IU penicillin and 100 ÎŒg/ml streptomycin (Wisent cat. number 450201).\n\nWestern Blot experiments were performed as described in our standard operating procedure.13 HAP1 WT and UBQLN2 KO were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat. number 0089901), supplemented with 1à protease inhibitor cocktail mix (MilliporeSigma, cat. number 78429). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000à g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and immunoblot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.\n\nWestern Blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer from bio-Rad (cat. number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat. number AAJ61337AD) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western Blots. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095) in TBS with 0.1% Tween 20 (TBST) (Cell Signalling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 ÎŒg/ml in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL (Thermo Fisher Scientific, cat. number 32106) prior to detection with the iBright⢠CL1500 Imaging System (Thermo Fisher Scientific, cat. number A44240).\n\nImmunoprecipitation experiments were performed as described in our standard operating procedure.14 Antibody-bead conjugates were prepared by adding 2 ÎŒg to 500 ÎŒl of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a 1.5 mL microcentrifuge tube, together with 30ÎŒl of Dynabeads protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively) or anti-Flag M2 magnetic beads from MilliporeSigma (cat. number M8823). Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.\n\nHAP1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) (Thermo Fisher Scientific, cat. number 87788) supplemented with protease inhibitor (Millipore Sigma, cat. number P8340). Lysates were rocked 30 min at 4°C and spun at 110,000à g for 15 min at 4°C. 0.5 ml aliquots at 2.0 mg/ml of lysate were incubated with an antibody-bead conjugate for ~1 hr at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP buffer and processed for SDS-PAGE and Western Blot on precast midi 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat number WXP42012BOX).\n\nImmunofluorescence was performed as described in our standard operating procedure.10 HAP1 WT and UBQLN2 KO were labelled with CellTrackerTM green (Thermo Fisher Scientific, cat. number C2925) or CellTrackerTM deep red (Thermo Fisher Scientific, cat. number C34565) fluorescence dye, respectively. The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. Number D3571) fluorescent stain. WT and KO cells were plated in 96 well glass plates (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37oC, 5% CO2. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS containing 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary Ubiquilin-2 antibodies overnight at 4°C. Cells were then washed 3 à 10 min with IF buffer and incubated with the corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 ÎŒg/ml for 1 hr at room temperature with DAPI. Cells were washed 3 à 10 min with IF buffer and once with PBS.\n\nImages were acquired on an ImageXpress micro widefield high-content microscopy system (Molecular Devices), using a 20Ã/0.45 NA air objective lens and scientific CMOS camera (16-bit, 1.97mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTrackerTM Green, Alexa fluor 555 and CellTrackerTM Red, respectively. Images had pixel sizes of 0.68 à 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTrackerTM channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the âcytoâ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns.15 Masks were used to generate cell outlines for intensity quantificationFigures were assembled with Adobe Illustrator.",
"appendix": "Data availability\n\nZenodo: Antibody Characterization Report for Ubiquilin-2, https://doi.org/10.5281/zenodo.7459541. 16\n\nZenodo: Dataset for the Ubiquilin-2 antibody screening study, https://doi.org/10.5281/zenodo.7671135. 17\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Ian McDowell, Peter S. McPherson, Chetan Raina, Wolfgang Reintsch and Kathleen Southern.\n\nABIF consortium: Claire M. Brown and Joel Ryan.\n\nAn earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.7459541).\n\n\nReferences\n\nKleijnen MF, Shih AH, Zhou P, et al.: The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome. Mol. Cell. 2000; 6(2): 409â419. PubMed Abstract | Publisher Full Text\n\nDeng HX, Chen W, Hong ST, et al.: Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Nature. 2011; 477(7363): 211â215. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHjerpe R, Bett JS, Keuss MJ, et al.: UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome. Cell. 2016; 166(4): 935â949. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPicher-Martel V, Dutta K, Phaneuf D, et al.: Ubiquilin-2 drives NF-κB activity and cytosolic TDP-43 aggregation in neuronal cells. Mol. Brain. 2015; 8(1): 71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPicher-Martel V, Renaud L, Bareil C, et al.: Neuronal Expression of UBQLN2(P497H) Exacerbates TDP-43 Pathology in TDP-43(G348C) Mice through Interaction with Ubiquitin. Mol. Neurobiol. 2019; 56(7): 4680â4696. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRenaud L, Picher-Martel V, Codron P, et al.: Key role of UBQLN2 in pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Acta Neuropathol. Commun. 2019; 7(1): 103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDao TP, Kolaitis RM, Kim HJ, et al.: Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions. Mol. Cell. 2018; 69(6): 965â78.e6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nAlshafie W, McPherson P, Laflamme C: Antibody screening by Immunofluorescence.2021.\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358âD367. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25â38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAyoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021.\n\nAyoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021.\n\nStringer C, Wang T, Michaelos M, et al.: Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021; 18(1): 100â106. Publisher Full Text\n\nMcDowell I, Fotouhi M, Ryan J, et al.: Antibody Characterization Report for Ubiquilin-2.2022. Publisher Full Text\n\nLaflamme C: Dataset for the Ubiquilin-2 antibody screening study. [Dataset]. Zenodo. 2023. Publisher Full Text"
}
|
[
{
"id": "173282",
"date": "26 May 2023",
"name": "Mervyn Monteiro",
"expertise": [
"Reviewer Expertise Neurodegenerative disorders"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an excellent study comparing the utility of different commercial UBQLN2 antibodies. The authors have tested the utility and specificity of these antibodies for Western blotting, immunoprecipitation and immunofluorescence staining using a UBQLN2 expressing and knockout cell lines. Readers will be able to determine which of the antibodies are specific for UBQLN2. One limitation of the approach is the choice of the cell line, which seems to express little UBQLN4. The lack of its expression makes it difficult to assess if the UBQLN2 antibodies cross-react with UBQLN4.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
},
{
"id": "173280",
"date": "08 Jun 2023",
"name": "Carlos Castaneda",
"expertise": [
"Reviewer Expertise biophysics",
"protein structure and function",
"biomolecular condensates",
"protein quality control",
"and ubiquitin biology."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis data note is a very important resource for the ubiquilin (UBQLN) community, as it does a very good job systematically comparing ten commercially available UBQLN2 antibodies. UBQLN2 is an important disease-linked protein whose functions are connected with the ubiquitin-proteasome system, autophagy, and stress-induced condensates. The study raises the important awareness that there are substantial differences in how these antibodies detect UBQLN2 in cell lysates, immunoprecipitation assays, and immunofluorescence experiments that examine subcellular localization of UBQLN2. The use of standardized controls and procedures enables a clear delineation as to which antibodies work best and which do not. It will be an excellent reference for many labs investigating UBQLN2 function in cells. There are only a couple of suggestions to further improve this note:\nInclude information (if available) on antibody epitope region; this will help ascertain whether antibodies potentially detect other related UBQLN paralogs (e.g. UBQLN1 or UBQLN4).\n\nIn the introduction, rephrase âand /or regulation of stress granulesâ.\n\nThe title should be reworded to say âhigh-performingâ (performing is misspelled).\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-355
|
https://f1000research.com/articles/12-354/v1
|
30 Mar 23
|
{
"type": "Research Article",
"title": "Predictors of chronic kidney disease among Indonesian adult population: Results from the 2018 Indonesia Basic Health Research",
"authors": [
"Qonita Rachmah",
"Dominikus Raditya Atmaka",
"Nila Reswari Haryana",
"Zulfitri Azuan Mat Daud",
"Qonita Rachmah",
"Nila Reswari Haryana",
"Zulfitri Azuan Mat Daud"
],
"abstract": "Background: Three out of ten cases of chronic kidney disease (CKD) are cause by type 2 diabetes mellitus (T2DM). In addition, nutritional status, consumption of unhealthy foods, high blood pressure, and smoking habits were also previously identified as risk factors to CKD but there is a scarcity of data from Indonesia. Therefore, we sought to determine the risk factors using a predictive model for the incidence of CKD in Indonesia based on the Indonesia Basic Health Research 2018. Methods: Secondary data from the 2018 Indonesia Basic Health Survey with 300,000 respondents was used. Respondents with complete socio-demographic, food consumption data, anthropometric data and chronic disease status (i.e. T2DM and hypertension) type 2 were analyzed (n=96,098). Binary logistic regression model was performed to assess the association between socio-demographic, food group consumption, health and nutritional status with CKD. Results: Â Based on the final model of logistic regression analysis, ranging from the most and least factors that contribute to CKD were the presence of T2DM (p<0.000; OR=2.353), hypertension (p<0.000; OR=1.695); education level (p=0.028; OR=1.438), living area (p=0.025; OR=1.242); age (p<0.000; OR=0.979); and sugary drink consumption (p=0.050; OR=0.884). Conclusions: Prediction equation of CKD risk factor in Indonesia Adult included 6 factors that are present: diabetes, present hypertension, education, living area, age, and sugary drink consumption. This result can be used by the health professional to predict the risk of CKD among patients with present diabetes and/or hypertension.",
"keywords": [
"chronic kidney disease",
"obesity",
"predictive model",
"Indonesia",
"adult"
],
"content": "Introduction\n\nDeaths due to non-communicable diseases have increased in the last decade and are predicted to increase to more than 380 million cases by 2025, especially in Southeast Asia, the Mediterranean and the Middle East and Africa. Chronic Kidney Diseases (CKD) is one of emerging public health problem that manifests by loss of kidney function, heightened cardiovascular diseases (CVD), and premature death.1,2 KDIGO in 2020 define CKD as an abnormalities of kidney function or anatomy structure with specific symptoms presents for more than three months and has implication for further health condition. While the burden of CKD is reasonably well defined in developed countries,1 increasing evidence indicates that the CKD burden may be even greater in developing countries.3 The 2014-2018 Indonesia National Health Insurance Statistics results show a substantial increase in the number of patients, the number of visits, the average number of visits per person/year and the cost of claims for dialysis procedures,4 inherent with the increasing financial burden for CKD treatment, with an average claim value per patient in a year as much as Rp. 48,714,515 or equivalent to USD 3,365.5\n\nIn Indonesia, the prevalence of CKD in 2013 was reported as 2%.6 In 2018, it was reported from the number of CKD patients, 19% received hemodialysis.7 Three out of 10 cases of CKD are caused by type 2 diabetes mellitus.8 In addition to diabetes, other conditions that are risk factors for CKD are nutritional status, consumption of unhealthy diets, high blood pressure, and smoking habits. In general, risk factors for CKD are divided into four main groups, namely susceptibility factors which are defined as a person's susceptibility factor to develop CKD, second is initiation factors including risk factors that can directly initiate kidney damage, then the progression factors include several factors that cause progressive kidney damage and accelerate the decline in kidney function; the last is end-stage factors which are factors that can increase morbidity and mortality due to CKD.9\n\nA prospective study on 23,543 respondents in Maryland, USA indicated that most of the risk of CKD in this population was associated with stage 1 hypertension (23%) and smoking (31%).10 The prevalence of CKD in diabetic patients is also increasing worldwide. CKD affects 50% of individuals with type 2 diabetes mellitus (T2DM).11 There is a strong correlation between the pathophysiology of kidney and heart disease in type 2 diabetes, this aspect is expressed by cardiovascular risk factors: T2DM, obesity, smoking, dyslipidaemia, hypertension, genetic factors, etc.12 Obesity is also a strong risk factor for kidney disease. Obesity increases the risk of major risk factors for CKD such as hypertension and diabetes. In obesity, the kidneys have to work harder to filter more blood to meet metabolic needs due to weight gain. This increase in function can damage the kidneys and increase the risk of developing CKD in the long term.13 Research on obese students at the Faculty of Medicine, Sam Ratulangi University, Indonesia revealed a strong relationship between body mass index (BMI) and a decrease in glomerular filtration rate (p < 0.001).14 Moreover, the prevalence of obesity in Indonesia has increased significantly from 2013 to 2018 from 26.3% to 35.4%. This can lead to an increase in the risk of CKD in Indonesian society and increase the financing burden.\n\nDifferent conditions in urban and rural areas can also affect differences in risk factors for CKD.15 This study aimed to determine the risk factors for CKD and derived a predictive equation for the incidence of CKD in Indonesia based on the results of basic health research 2018 that represents each region in Indonesia. This analysis is expected to provide significant results and have an impact on appropriate CKD prevention efforts in Indonesia. CKD prevention efforts as early as possible can have a positive impact, not only maintaining productivity, maintaining quality of life, reducing morbidity and mortality but also reducing the burden of disease due to CKD.\n\n\nMethods\n\nThis was a cross-sectional study which used secondary data from the 2018 cohort Indonesia Basic Health Survey (RISKESDAS) upon request and approval from the National Institute of Health Research and Development, Ministry of Health, Republic of Indonesia with a total number of respondents of 300,000. RISKEDAS is a national representative household health survey conducted every three years by the Indonesia National Institute of Health Research and Development (NIHRD), Ministry of Health Indonesia. Data for Riskesdas were collected by qualified enumerator that collected from representative areas of Indonesia, both rural and urban. Respondents who provided sociodemographic, food consumption, type 2 diabetes status (self-reported), hypertension status (self-reported), and body mass index data were further analyzed. We excluded respondents who were aged <18 years to minimize the sampling bias. Further, we included only respondents who had no missing data. After the inclusion criteria were applied, 96,098 respondents were included in this study.\n\nDiagnosis of CKD was noted by the self-reported declaration on whether any physicians ever diagnosed and informed them that they had CKD with symptoms persists for a minimum of three months based on the result of glomerulus filtration rate (GFR).7\n\nSociodemographic characteristics including marital status, level of education, geographical areas of living, sex, and working status were reported using categorical data with the exception for age, accessed using ratio data. The respondentsâ marital status was categorized into never been married, married, and divorced. The respondentsâ level of education was categorized into low (<6 years of school attainment), middle (6-12 years of school attainment) and high (>12 years of school attainment). Living area was categorized into urban and rural; while working status was classified as working and not working.\n\nWe also obtained information on T2DM and hypertension as the risk factors of CKD. Respondents answered the self-reported questionnaire on whether they have been diagnosed with T2DM and hypertension.7 The body mass index (in kg/m2) was classified into four groups (underweight <18.5, normal 18.5â25.0, overweight 25.1â27.0, and obese >27.0).16 In addition, food group consumption was measured using a Food Frequency Questionnaire (FFQ). Respondents were asked the frequency of consumption of non-healthy foods in a day, week or month including consumption of sugary foods and beverages, salty foods, fatty/fried foods, grilled foods, processed meats, seasonings, soft-drinks or carbonated drinks, energy drinks, and instant noodles.\n\nThe respondentsâ sociodemographic data were presented as means (standard deviation) for the continuous data and numbers (percentages) for the categorical data. For the bivariate analysis, chi-square test was used to analyse the relation between independent and dependent variables in categorical data; while t-test analysis was used for continuous data. Moreover, we used a binary logistic regression model to assess the association between socio-demographic, food group consumption, health and nutritional status with CKD. All the analyses were conducted using SPSS statistical software (V 26; SPSS Inc., IBM Corporation).\n\n\nResults\n\nAmong 96,098 adults were involved in this study, 0.5% or 501 adults had CKD (Table 1). There was a significant (p < 0.001; 2.000-2.003% CI) difference in age between adults with CKD and non-CKD. The older adults group had a 2.002 times higher risk of getting CKD than the younger adults group. So that age factor was one of the greatest risk among other factors.\n\n* p < 0.05.\n\n** p < 0.01.\n\n*** p < 0.001. P was obtained with Ï2 tests.\n\n+ Analyze using T-test.\n\nA total of 54,844 respondents lived in rural and had significantly less risk (p < 0.05; 0.665-0.956% CI) of 0.798 times lower than respondents who lived in urban. In other words, adults who lived in an urban area had a 1.25 times higher risk to get CKD than adults who lived in rural areas. Meanwhile, sex was not identified as a risk factor of CKD (p > 0.005), and yet, the majority of respondents in this study were male (94.6%). The majority of respondents were married. Interestingly, marital status was identified as one of the risk factors of CKD (p < 0.001; 0.498-0765% CI), in which unmarried adults had 1.62 times greater risk of developing CKD than their married counterparts. Most of the respondents in the study had a low education status in which they never attended a formal school or only completed elementary school. Lower educational background posts a risk towards CKD (p < 0.05; OR 1.229; 1.061-1.425% CI). There was significant difference between adults with CKD and non-CKD with regards to their employment status (p < 0.01; OR:1.438; 1.099-1.881% CI) in which a higher risk was observed for working adults (1.438 times) than the non-working adults.\n\nIn total, five dietary patterns from total of 10 food groups were identified to be significantly different between adults with and without CKD (p < 0.05) (Table 2). Five dietary patterns that are associated with CKD are consumption of sugary foods, sugary drinks, soft drink, energy drinks, and instant noodles. Compared to other dietary patterns, instant noodles have the highest significant differences between adults with and without CKD (p = 0.000) which means the consumption of instant noodle give higher impact to the development of CKD. An individual with high consumption of sugary foods has 1.041 times higher risk to develop CKD compared to those with low consumption of sugary foods (less than 3 times per months or never consume sugary foods). Meanwhile high consumption of sugary drinks, soft drinks, energy drinks, and instant noodles can lead to a higher risk of developing CKD compared to those who consume low amounts of sugary drinks, soft drinks, energy drinks, and instant noodles with OR respectively 1.175; 1.004; 1.804; and 1.202.\n\n* p < 0.05.\n\n** p < 0.01.\n\n*** p < 0.001. P was obtained with Ï2 tests.\n\nThe present of comorbidities particularly T2DM and hypertension were associated with CKD (p < 0.000) (Table 3); but not nutritional status (p > 0.05). An individual with type 2 diabetes mellitus has 3.240 times higher risk of developing CKD compared to those without diabetes. While hypertension increased the risk of CKD by 2.286 folds. Further logistic regression analysis revealed the Hosmer value was 0.088; this means that the logistic regression equation can be used to explain the relationship between the independent variable and the dependent variable. Based on the final model of logistic regression analysis, ranging from the factors contributing the most to the least to CKD were the presence of T2DM (p < 0.000; OR = 2.353; 1.625-3.405 95% CI), presence of hypertension (p < 0.000; OR = 1.695; 1.346-2.133 95% CI); education (p = 0.028; OR = 1.438; 1.039-1.989 95% CI), living area (p = 0.025; OR = 1.242; 1.028-1.500 95% CI); age (p < 0.000; OR = 0.979; 0.972-0.987 95% CI); and sugary drink consumption (p = 0.050; OR = 0.884; 0.781-1.000 95% CI).\n\n* p < 0.05.\n\n** p < 0.01.\n\n*** p < 0.001. P was obtained with Ï2 tests.\n\n# T2DM and hypertension data was based on self-reported data.\n\nFinally, the CKD risk prediction equation resulting from this study is: 3,746 + 2.353 (present of T2DM) +1.695 (present hypertension) + 1.438 (education) + 1.242 (living area) + 0.979*age + 0.884 (sugary drink consumption). Coding for each variable is as follow: present of T2DM (1: present; 0: not present); present of hypertension (1: present; 0: not present); education (1: not school/not graduate elementary/elementary; 0: middle to high education); living area (1: rural; 0: urban); and sugary drinks consumption (1: 1Ã/day or more; 0: weekly/monthly/never).\n\n\nDiscussion\n\nOlder age was reported to be associated with increased risk of reduced renal function and they have high prevalence of more severe stages of CKD in elderly individuals.17,18 Increasing age affects the anatomy, physiology and cytology of the kidneys. Decreasing the estimated glomerular filtration rate (eGFR) is a ânormal agingâ process. The kidneys cannot regenerate new nephrons, so that if the kidneys are damaged or in the aging process, there will be a decrease in the number of nephrons. After the age of 40, the number of functioning nephrons decreases by about 10% every 10 years and after the age of 80 the kidneys have only 40% of the functioning.19â21 The cohort of the Framingham Offspring Study in 2,585 subjects without CKD followed for 12 years, showed an age-related decrease in GFR (OR = 2.36 per 10-year increase in age; 95% CI 2.00-2.78)22 concurred with the findings from our study that age is the biggest predictor of CKD.\n\nThe lifestyle of urban adults has experienced adaptation and sedentary. This is caused by the type and work environment, population density in settlements and the influence of the media, especially television.23 In India, urbanisation is associated with risk factor of non-communicable diseases.24 Compared with their rural counterparts, men working in factories have approximately 1.5-fold increase in odds of a sedentary lifestyle, three-fold increase in odds of obesity, and two-fold increase in odds of T2DM.25 The results of this study is in line with Singh et al.26 that showed that patients with CKD were older, more likely to be male, more likely to have a high school diploma, more likely to reside in urban areas, less likely to have a low income, more likely to be overweight or obese, to present with comorbidities including diabetes, hypertension and cardiovascular disease than patients without CKD.\n\nOne of the important factors in determining health status is educational background. The higher the level of education, the higher the awareness of the importance of health.27 Different education level will affect level of knowledge and it causes differences in responses to a health problem. In addition, there will be different levels of understanding of information that conveyed about the disease suffered. People with low educational background tend to have awareness to do early detection of CKD. This can increase the awareness of patients with CKD because in the early stages the signs and symptoms are not felt specifically. Most of the patients came with severe complaints and at the time of further examination were already at the end stage (stage 5).28\n\nBeing single or divorced/widowed increased the OR 3 fold compared to those who are married. Additionally in sex stratified analysis among women, being single or divorced/widowed relative to being married were positive predictors for incident of CKD stages 3 to 5.29 This is in line with the study by Novak et al.30 that showed that people with depression had a significantly higher risk of developing CKD and those who depressed were younger, more likely to be divorced and had higher eGFR. Thus, it is postulated that someone who is single/divorced has potential to experience stress, inherit to increase risk of other chronic diseases such as hypertension, T2DM that can lead to chronic kidney failure.31 Most people with CKD have a lower quality of life than non-CKD people.32 Most patients cannot return to their previous activities or work. Decreased body function and limitations in carrying out activities are the cause, so they prefer to rest and not work.33\n\nDietary pattern is reflected the habitual dietary intake which can be classified into healthy and unhealthy pattern. Meta-analysis by Bach et al. show intake of low red and processed meats, sodium, also sugar sweetened foods associated with lower odds ratio of CKD incident.34 Meanwhile high consumption of refined grains, high fat dairy foods, meat, beans highly associated with albumin to creatinine ratio which give higher impact in kidney injury.35\n\nConsumption of several unhealthy foods such as added fats, sugars, salt, and refined grains are associated with adverse metabolic outcome, including CKD.36,37 Excess calorie intake is an underlying factor of the development of obesity which may give consequences for long term kidney health. The higher calorie intake, the higher body mass index, also the higher prevalence of kidney injury and increase risk of CKD incident.38â44\n\nConsumption of drinks containing high amounts of sugar especially fructose (sugar cane based sugar, the most used kind in Indonesia) is associated with the increasement risk of kidney disease.45 High consumption of refined sugar may increase triglycerides level and increase body mass index. The elevation of blood glucose also give consequences in kidney failure by metabolic impairment like induce insulin resistance and produce more uric acid.46 Besides, polyol pathway from glucose to fructose conversion can cause kidney damage.47 This study has the same result as a study in Iran which showed high fat and sugar food and drink choices significantly associated with the increased of incident risk of CKD by 46%.48 Consumption of artificial sweetened soda showed association with incidence of albuminuria and rapid GFR decline.49,50 In addition, dietary sugar increases the risk of diabetes mellitus which being the high risk factor of CKD.45\n\nOne of the findings in this study is that consumption of instant noodles, which are high in sodium, is significantly higher in adults with CKD. More than 75% of salt intake are coming from processed foods, one of which is instant noodle.51 Excess salt intake is associated with adverse health outcome like hypertension, volume overload, vascular damage and ventricular hypertrophy, which exacerbate CKD. The higher sodium intake, the higher the blood pressure, external fluid volume, and albuminuria which may be implicated in CKD incidence.52â56 Excess sodium intake also increases kidney plasma flow and glomerular filtration pressure which contributes to proteinuria and faster progression to kidney failure.57,58\n\nThe association between T2DM and CKD as well as hypertension and CKD has been long known both as initiation factors or risk factors that can directly initiate kidney damage. KDOQI statement also mention that diabetes as marked by hyperglycemia which causes vascular complication and diabetic kidney disease (DKD).59 A prospective study, NHANES, from 2009-2014 in the US revealed that CKD prevalence was progressively higher with longer duration of diabetes. Further, they explained that increase risk of CKD among people with diabetes could be mediated by hyperglycemia-induced glomerulopathy, vascular damage, treatment specific to diabetes, or other confounding such as smoking, obesity or genetic predisposition.60 Similarly, Szczech, et al. (2014) also reported that presence of diabetes ultimately increases CKD risks by modifying urine protein excretion and decreased eGFR.61 Moreover, mitochondrial dysfunction among TD2M individual plays an important role in the existence of diabetic complication through increasement of reactive oxygen species (ROS) and superoxide generation.62,63\n\nKim et al. (2014) proposed that the risk of kidney damage in T2DM could be induced by the higher level of serum uric acid.64 High serum uric acid increased renin-angiotensin-aldosterone (RAA) system and inflammation, also dysfunction the endothelial. Those pathways were similar to the pathway of hypertension to CKD. Hypertension and CKD have an inverse relationship, untreated hypertension increases the risk of CKD and CKD can also worsen the condition of hypertension to cause cardiovascular complications.65,66 In all types of glomerulonephritis, hypertension is a substantial independent risk factor for progression to End-Stage Renal Diseases (ESRD). Therefore, therapeutic intervention is highly needed in hypertensive patient to prevent the kidney damage.67\n\nOur results did not find a significant correlation between weight status and CKD, but were significant with the presence of T2DM and hypertension. We suggest these results could imply that CKD might present to those with previously diagnosed T2DM or hypertension without the individual being overweight/obese. However, it is well-known that obesity is a major risk factor of metabolic disorders such as TD2M and hypertension that lead to increased risk of kidney failure.68 Impaired pressure natriuresis and increase of tubular sodium reabsorption affects volume expansion through sympathetic nervous system then activate RAA-system; especially with the presence of visceral obesity, in the end, raises blood pressure. In addition, increased oxidative stress, inflammation, and lipotoxicity also increases the risk of kidney damage among obese hypertensive.68,69\n\nTo the best of our knowledge, there were no previous studies in Indonesia which had reported predictors of CKD among Indonesian adults, which makes this report novel. Despite a cross-sectional nature, our study analyzed a huge number of samples which we believe can be generalized to the Indonesian adult population. However, we also note some limitations such as self-declaration of prior diagnosis of CKD, T2DM and hypertension that may undermine undiagnosed cases and memory bias. Despite the presence of aforementioned bias, there was no other similar study in Indonesia therefore the novelty of this study is accountable. In future research, database should be compared from year to year included more risk factors that might correlated with CKD to give better perspective on CKD trend in Indonesia.\n\n\nConclusion\n\nThis result can be used by the health professional to predict the risk of CKD based on the identified risk factors. Early prediction of CKD risk could be beneficial to prevent the development of CKD by changing dietary and lifestyle behavior; thus, prolonging the need for dialysis treatment. Due to the data limitations of the study, for further studies we suggest researchers can identify specific factors influencing the occurrence of kidney diseases in the Indonesian population.",
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PubMed Abstract | Publisher Full Text\n\nMcMahon EJ, Bauer JD, Hawley CM, et al.: A randomized trial of dietary sodium restriction in CKD. J. Am. Soc. Nephrol. 2013; 24(12): 2096â2103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSanders PW: Vascular consequences of dietary salt intake. Am. J. Physiol. Renal Physiol. 2009; 297(2): F237âF243. Publisher Full Text\n\nKrikken JA, Laverman GU, Navis G: Benefits of dietary sodium restriction in the management of chronic kidney disease. Curr. Opin. Nephrol. Hypertens. 2009; 18(6): 531â538. Publisher Full Text\n\nRitz E: Salt - Friend or foe? Nephrol. Dial. Transplant. 2006; 21(8): 2052â2056. Publisher Full Text\n\nRocco MV, Berns JS: KDOQI clinical practice guideline for diabetes and CKD: 2012 update. Am. J. Kidney Dis. 2012; 60(5): 850â886. Publisher Full Text\n\nZelnick LR, Weiss NS, Kestenbaum BR, et al.: Diabetes and CKD in the United States population, 2009â2014. Clin. J. Am. Soc. Nephrol. 2017; 12(12): 1984â1990. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzczech LA, Stewart RC, Su HL, et al.: Primary care detection of chronic kidney disease in adults with Type-2 diabetes: The ADD-CKD study (awareness, detection and drug therapy in type 2 diabetes and chronic kidney disease). PLoS One. 2014; 9(11): 1â16. Publisher Full Text\n\nReidy K, Kang HM, Hostetter T, et al.: Molecular mechanisms of Diabetic kidney disease. J. Clin. Invest. 2014; 124(6): 2333â2340. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrownlee M: The pathobiology of diabetic complications: A unifying mechanism. Diabetes. 2005; 54(6): 1615â1625. Publisher Full Text\n\nKim WJ, Kim SS, Bae MJ, et al.: High-normal serum uric acid predicts the development of chronic kidney disease in patients with type 2 diabetes mellitus and preserved kidney function. J. Diabetes Complicat. 2014; 28(2): 130â134. PubMed Abstract | Publisher Full Text\n\nKu E, Lee BJ, Wei J, et al.: Hypertension in CKD: Core Curriculum 2019. Am. J. Kidney Dis. 2019; 74(1): 120â131. PubMed Abstract | Publisher Full Text\n\nKuriyama S, Maruyama Y, Nishio S, et al.: Serum uric acid and the incidence of CKD and hypertension. Clin. Exp. Nephrol. 2015; 19(6): 1127â1134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMennuni S, Rubattu S, Pierelli G, et al.: Hypertension and kidneys: Unraveling complex molecular mechanisms underlying hypertensive renal damage. J. Hum. Hypertens. 2014; 28(2): 74â79. PubMed Abstract | Publisher Full Text\n\nHall JE, Henegar JR, Dwyer TM, et al.: Is Obesity a Major Cause of Chronic Kidney Disease? Adv. Ren. Replace. Ther. 2004; 11(1): 41â54. PubMed Abstract | Publisher Full Text\n\nHall JE, Crook ED, Jones DW, et al.: Mechanisms of obesity-associated cardiovascular and renal disease. Am. J. Med. Sci. 2002; 324(3): 127â137. Publisher Full Text"
}
|
[
{
"id": "248200",
"date": "14 May 2024",
"name": "Bayu Satria Wiratama",
"expertise": [
"Reviewer Expertise epidemiology",
"road injury",
"non communicable disease",
"communicable disease",
"outbreak"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank your for the invitation to review this manuscript. This is a good manuscript but there are several queries need to resolved to improve the quality of the manuscript.\nMethods 1. The authors mentioned that \"Respondents who provided sociodemographic, food consumption, type 2 diabetes status (self-reported), hypertension status (self-reported), and body mass index data were further analyzed\" so for CKD variables, is it not considered? I suggest adding CKD variables in this sentences. 2. Authors mentioned that only include respondents with no missing data, is it for all variables? If authors decide to remove participants with missing data, please create a data flowchart explaining how many missing data for each variable and how many participants removed. 3. If the missing data reaches more than 5â10%, please also check whether there is a pattern of missing data and decide if the missing data is missing at random or not. 4. For age variable, is there any reason for using it as continuous instead of categorizing it into age group which has more meaningful interpretation (such as 18-45, 45-60, etc.)? 5. What is the category for geographical area of living variable? 6. Authors used hosmer lemeshow test in result but there is no explanation about it in the methods. Please add the explanation about hosmer lemeshow test and why authors used it instead of AIC/BIC? Because if we want to compare different model in multivariate then it is preferred to used AIC/BIC rather than hosmer lemeshow test. 7. Independent t-test is used if the outcome is a continuous data and independent variable is category but in this manuscript the other way around. So it will be better if author used a simple binary logistic regression to test the relationship between age and CKD while also obtain the crude OR.\nResult 8. Authors using risk in all of the result section but this is a logistic regression result which use odds instead of risk. Please give explanation in methods about the use of risk instead of odds using logistic regression. Because if we decide to use risk then we will use risk ratio as the effect size and modified poisson or cox regression will be used as the multivariate analysis. 9. Authors mentioned that in the final model, rural area was associated with increased 24% odds of CKD but in the discussion authors said that patients with CKD more likely to reside in urban area. please double check and revise this accordingly. 10. Authors mentioned that there were no previous studies about predictors of CKD but there are several article about risk factors of CKD in Indonesia:  (Hidayangsih PS, et al., 2023 [Ref 1]), (Hustrini NM, et al., 2022 [Ref 2]) Both of this article use the same dataset. Please explain about this and what is the difference and strength of your manuscript compared with previous articles. 11. Conclusion must start to answer the aim of this study mentioned in introduction which is the risk factors/predictors of CKD then about the risk prediction model. Please revise the conclusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "232573",
"date": "28 Aug 2024",
"name": "Mahulul Azam",
"expertise": [
"Reviewer Expertise Epidemiology",
"public health",
"preventive medicine"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study's strength was that it presented data from the national scope in Indonesia regarding the factors related to CKD in adult populations.\nSome points that must be revised to improve the readability included - to  fix grammatical errors (in my opinion, there are some errors, but it is not my capacity to review this); - in the title, the authors use the Indonesia basic health research term, but in the manuscript, the term uses Indonesia Basic Health Survey; please use the term consistently and commonly in other reports. - in the background the statements In general, risk factors for CKD are divided into four main groups, namely susceptibility factors which are defined as a person's susceptibility factor to develop CKD, second is initiation factors including risk factors that can directly initiate kidney damage, then the progression factors include several factors that cause progressive kidney damage and accelerate the decline in kidney function; the last is end-stage factors which are factors that can increase morbidity and mortality due to CKD. I didnât find these factors in this reference, or you might show me this statement in this reference (The impact of social support on end-stage renal disease) as you cited. - in methods, please add the statement that this study conformed the ethical procedure, refer to the approval of ethical clearance in the Riskesdas survey - please add explanation for the certain term that might be a little bit confused for readers like energy drinks, and other terms that needed. - In table 1, I encourage to provide the table 1 as a cross sectional study with 1 column proportion instead of two column as CKD and non CKD that commonly used in a case control study, this table can be provided as table 2 (table 2a, 2b etc if so many parameters must be presented) - discussion regarding the number of prevalence i.e. 0.5 %, in my opinion is also need to be much more explain and comparing with other natonal studies around the world, since this is the strengh of this study - this predictor model build form a cross sectional study that have a limitation that can not explain the exposure occurred before the effect, I think author can provide this limitation in the discussion as well as a limitation of absence the detailed data of the duration of hypertension and diabetes mellitus that known as predictor/ main risk factors of CKD.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-354
|
https://f1000research.com/articles/12-353/v1
|
30 Mar 23
|
{
"type": "Research Article",
"title": "Fixed dose combination of low dose pregabalin and duloxetine, or pregabalin monotherapy for neuropathic pain: A double-blind, randomized, parallel-group study",
"authors": [
"Krishnaprasad K.",
"Sunil Dutt",
"Pankaj Rattan",
"Ankit Dadhania",
"Ram Gupta",
"Deepa Joshi",
"Ashutosh Kakkad",
"Altaf Makwana",
"Pankaj Jha",
"Sunil Dutt",
"Pankaj Rattan",
"Ankit Dadhania",
"Ram Gupta",
"Deepa Joshi",
"Ashutosh Kakkad",
"Altaf Makwana",
"Pankaj Jha"
],
"abstract": "Background: Treatment of neuropathic pain is challenging. Pregabalin and duloxetine are used as first-line therapy. Various international guidelines recommend a combination of first-line agents for the management of neuropathic pain. The objective of this study was to evaluate the efficacy and safety of a fixed-dose combination (FDC) of low-dose pregabalin and duloxetine compared to pregabalin monotherapy at week 7 in patients with moderate to severe neuropathic pain. Methods: This was a phase 3, randomized, double-blind, double-dummy parallel-group non-inferiority study conducted at 17 sites across India. Three hundred and twenty-eight adult patients with moderate to severe neuropathic pain were randomized in a ratio of 1:1 to receive a FDC of pregabalin and duloxetine or pregabalin monotherapy for 7 weeks followed by a one-week follow-up. The pregabalin-duloxetine combination was initiated at 50 plus 20 mg per day and gradually titrated to a maximum of 75mg plus 30mg twice daily. Pregabalin was initiated at 75mg/day and gradually titrated to a maximum of 150mg twice daily. The main efficacy outcome was a mean change in pain intensity at the end of 7 weeks. Results: Two hundred and ninety-eight patients completed the study, 148 in the pregabalin-duloxetine group and 150 in the pregabalin group. The mean change in daily pain at 7 weeks was as follows: -4.49 with FDC and -4.66 with pregabalin (p<0.0001). The non-inferiority of a low-dose FDC compared to pregabalin monotherapy was demonstrated at the end of the study. The incidence of dizziness and somnolence was comparable between both treatments. A higher frequency of peripheral oedema was observed with pregabalin monotherapy than in the FDC group (p>0.05). Conclusions: A FDC of low doses of pregabalin and duloxetine and high dose of pregabalin monotherapy achieved similar analgesia with dizziness, and somnolence as the most frequent adverse event. Trial registration: CTRI/2020/09/027555",
"keywords": [
"Pregabalin",
"duloxetine",
"fixed-dose combination",
"neuropathic",
"pain",
"numeric pain rating scale (NPRS)"
],
"content": "Introduction\n\nNeuropathic pain significantly impacts the quality of life and well-being of patient.1 The financial burden to manage neuropathic pain in the chronic setting is huge on society.2,3 Its prevalence in the general population ranges from 1% to 8%.4,5\n\nThe most effective pharmacotherapeutic classes of drugs in the management of neuropathic pain are gabapentanoids, tricyclic antidepressants (TCAs) and serotonin-norepinephrine reuptake inhibitors (SNRIs).1â3,6â9 Most of these options are effective but come with a few potential side effects.\n\nPregabalin, a gabapentanoid blocks the presynaptic alpha 2 delta subunit of calcium channels of the dorsal horn. This leads to inhibition of neurotransmitter release from presynaptic neuron.7,8,10 Multiple international societies recommend it as a first-line treatment option for neuropathic pain.2,6,11\n\nSNRIs are also recommended as a first-line treatment option by multiple guidelines.1â3,6â9 Duloxetine, an SNRI modulates pain signals by facilitating descending inhibitory pain pathway.3,7 SNRIs have been found effective in peripheral diabetic neuropathy,6,8 painful peripheral neuropathy and multiple sclerosis.12\n\nTreatment of neuropathic pain with monotherapy has several limitations such as not being effective for all patients, having partial pain relief and increased side effects with higher doses.8 These are the main reasons to use two or more drugs in about 45% of patients.13,14 To overcome these issues, for adequate pain relief without increased adverse effects, combination therapy with two or more medications is required in about half of the patients affected with neuropathic pain. Many international guidelines suggest an addition of another drug from a different pharmacological class when adequate pain control is not achieved with monotherapy. However, the evidence available for combination therapy is limited.1,2,7,8,9 The advantages of combination therapy may be increased efficacy and better tolerability due to the use of smaller doses of individual drugs.1,9\n\nBoth pregabalin and duloxetine are approved in the treatment of neuropathic pain. The monotherapy with drugs at their standard doses (60mg and 300 mg/day respectively) have shown to provide substantial clinical pain relief in only 40% of patients.15,16,17\n\nAdministration of pregabalin and duloxetine combination treatment may provide better pain relief and tolerability than the administration of maximum doses of each drug, especially in patients showing partial response to monotherapy.18\n\nPregabalin and duloxetine have different mechanism of action and administration of these drugs in combination rather than monotherapy may potentially provide enhanced pain relief in neuropathic pain due to their complementary actions.19,20 However, there have been no clinical studies on fixed-dose combinations of low-dose pregabalin and duloxetine for the treatment of neuropathic pain.\n\nThe objective of our phase 3 study was to assess the potential non-inferiority of fixed-dose combination of low-dose pregabalin and duloxetine compared to pregabalin monotherapy in adults with moderate to severe neuropathic pain.\n\n\nMethods\n\nThis was a multicentre, parallel, randomized, double-blind, double-dummy, non-inferiority trial conducted in adult patients with neuropathic pain enrolled from 17 sites across India. All investigational sites were approved by respective Independent Ethics Committee or Institutional Review Board before the enrolment of any patient in the trial. This trial followed the principles outlined in the Declaration of Helsinki and the trial protocol was approved by the Institutional Ethics Committee and the Drug Controller General of India (CT-06-11/2020). The trial was registered on 2nd September 2020 with the Clinical Trial Registry of India (Registry identifier: CTRI/2020/09/027555). This trial was conducted from September 2020 to January 2021.\n\nFour hundred and thirty patients presenting with moderate to severe neuropathic pain and having at least moderate to severe pain intensity ⥠4 on the numeric pain rating scale (NPRS) during screening at one of the participating centres were assessed and referred to the investigator for enrolment. The pain intensity was measured through 11-point NPRS in which 0 indicated no pain and 10 as the worst possible pain. Eligible adult patients aged 18 to 65 years of age who met inclusion criteria and signed informed consent forms were enrolled. Exclusion criteria included were a history of hypersensitivity to pregabalin or duloxetine or any of its excipients; clinically significant illness in the past three months; type I or II diabetes with glycosylated haemoglobin A1C of >10%; clinically significant electrocardiogram (ECG) abnormalities or QTc â¥450 msec; treated with drugs that impair metabolism of serotonin or serotonergic drugs other than study drugs or corticosteroids within the past four weeks; treated with topical or systemic pain medications within past two weeks; pregnant or lactating women, female with childbearing potential who were neither surgically sterilized nor willing to use medically acceptable contraceptive methods during the study; participated in any other investigational drug trial within the past four weeks and those patients who in the opinion of the investigator either unable to cooperate or unlikely to adhere with any study procedures or not considered a suitable candidate for participation in the study.\n\nThe trial period was 7 weeks, and the patients were scheduled for 7 visits (screening, baseline, week 1, week 2, week 3, week 5 and week 7). Patients were screened by the respective site investigator and eligible patients were randomized to receive either test + dummy reference or reference + dummy treatment. The assignment of treatment was as per the pre-determined computer generated, centrally administered randomization scheduled with the help of interactive web response system (IWRS). Each patient received unique randomization code.\n\nPost-screening, three hundred and twenty-eight participants were randomized to receive either fixed-dose combination (FDC) of pregabalin and duloxetine or pregabalin monotherapy. At screening, sociodemographic data and pain intensity were assessed. At baseline, clinical data (significant medical history, concomitant use of other medications), pain intensity and neuropathic pain symptom inventory were collected.\n\nAt the baseline visit, all the eligible patients were randomized in a 1:1 fashion to receive either FDC of pregabalin and duloxetine plus dummy of pregabalin (Test group) or pregabalin plus dummy of FDC of pregabalin and duloxetine (Reference group). The study medications were administered in a double-blind, dummy manner. The double-dummy design of the trial was achieved with the placebo formulations of pregabalin plus duloxetine and pregabalin in identical capsule. The investigation productâs primary packaging, blinding, labelling and secondary packaging was performed by third party. The investigation packages were given unique number code. At regular monitoring visits, study monitor ensured that the blinding was maintained. The participants, the research staff, and the investigators were all blinded to treatment allocations throughout the study period. One-hundred and sixty-four patients in the test group were initiated with FDC of pregabalin 50mg plus duloxetine 20mg capsule and one-hundred and sixty-four patients in the reference group with pregabalin monotherapy (75mg) capsule in the evening. Patients who responded to assigned treatment continued the same treatment until week 7 and those who were non-responders were up titrated after assessment at consecutive weeks. Patients who were having <30% reduction in pain intensity (based on the 11-point NPRS) were considered non-responders to the assigned treatment. After visit 2, non-responders in both groups were up-titrated at visit 3, visit 4 and visit 5 (Table 1). A telephonic safety follow-up was performed for all the subjects after 1 week (± 2 days) after the end of the treatment.\n\n\n\n⢠Visit 3-75 mg plus 20 mg in the evening\n\n⢠Visit 4-75 mg plus 30 mg in the evening\n\n⢠Visit 5-75 mg plus 30 mg in the morning and evening\n\n\n\n⢠Visit 3-100 mg in the evening\n\n⢠Visit 4-150 mg in the evening\n\n⢠Visit 5-150 mg in the morning and evening\n\nThe primary objective was to assess the mean change in pain intensity based on an 11-point NPRS.\n\nThe secondary outcome measures were mean change in NPRS from baseline to weeks 2, 3, and 5; proportion of responders (â¥30% improvement in pain (NPRS) from baseline) at week 7; proportion of patients with â¥50% improvement in NPRS from baseline to week 7; mean change in NPSI total score from baseline to end of the treatment; Percentage of patients who required rescue medication for inadequate pain relief (recorded at every visit); Patient Global Impression of Improvement (PGI-I) score at week 7; Clinical Global Impression of Improvement (CGI-I) score at week 7; incidence of treatment-emergent adverse event (TEAE) in test arm against reference arm (recorded at every visit)\n\nThe study was powered to detect a mean of improvement of 2.5 for Pregabalin and 2.7 for FDC at the end of the study in NPRS with a 2-sided test and 95% power, assuming an SD of 2.3. Non-inferiority margin considered was 33%. This analysis required 139 patients per treatment arm. We assumed around 15% discontinuing during therapy, on that basis a total of 328 patients were planned.\n\nFor analyses, we used modified intention-to-treat (mITT) principles. All randomised patients were included in efficacy analyses if they were treated and had a baseline and at least received one dose of study medication and one post-baseline assessment of any efficacy parameter. In safety population analysis, all randomised patients who received at least one dose of study medication were included.\n\nPrimary efficacy evaluation, change from baseline (Day 0) to EOT (Week 7) was calculated for NPRS score. It was compared using an independent t-test to test the differences between treatment groups. The non-inferiority was assessed based on the one-sided 97.5% CI (i.e., two-sided 95% CI) approach. A non-inferiority margin of 33% (MoNI -0.8) of the total response was observed for reference.\n\nThe secondary efficacy analysis included mean change in NPRS score from baseline to Weeks 2, 3 and 5 and mean change in NPSI total score from baseline to end of week 7, PGI-I and CGI-I score, subjects required rescue medication for inadequate pain relief and incidence of TEAE [dizziness, somnolence and/or peripheral oedema]. These endpoints were analyzed using either t-test, Chi-square or Fisherâs exact test.\n\nDescriptive statistics were used to summarise the safety measures. Fisherâs Exploratory Analysis were performed to determine the frequencies of patients with TEAEs. The analysis of variance (ANOVA) and analysis of covariance (ANCOVA) were performed to determine the difference in the least-squares mean in the magnitude of change in NPRS and NPSI scores between the treatments. All statistical analysis were performed by using SAS software, version 9.4 (SAS Institute Inc., North Carolina, Cary, USA).\n\n\nResults\n\nA total of 430 patients were screened from all the sites (Figure 1). A total of 328 eligible patients were equally randomized in two groups as test groups (N=164) and reference group (N=164). After randomization, 20 patients withdrew their consent, 7 patients were lost to follow-up, 2 patients discontinued due to adverse events and 1 patient was discontinued by the investigator from the test group. The reasons for discontinuation are mentioned in Figure 1. Thus, total 298 patients completed 7 weeks treatment.28\n\nThe demographic characteristics between both treatment groups were comparable. The number of female patients was slightly higher in the reference group compared to the test group. The demographic characteristics are presented in Table 2. The mean patient age at enrolment was 46.5 years. All included patients had an average pain score of at least 4 on NPRS prior to treatment. The mean pain intensity was 6.4.\n\nThe primary efficacy assessment was to compare the change in NPRS score between the test and the reference group. At the end of week 7, the mean change in NPRS score from baseline was -4.49 and -4.66 with the test and the reference group (p<0.0001) respectively (Figure 2). The lower bound 95% CI for treatment difference was -0.18 which was higher than the non-inferiority margin of -0.8. Therefore, the test treatment was non-inferior to the reference group. These results obtained in the mITT population were supported by those seen in the PP population [95%CI: -0.26, 0.44, p <0.05).\n\nThe change in NPRS scores between the test group and the reference group at weeks 2, 3 and 5 from baseline was non-significant. In the test group it was -2.49, -3.19 and -3.89 while -2.65, -3.34 and -4.03 in the reference group (p>0.05). (Figure 2)\n\nThe percentage proportion of responders between the test and the reference treatment were similar at week 7 (96.20% Vs 98.73%, p>0.05). Similarly, no statistically significant difference was observed at week 7 in the percentage of the patient with â¥50% improvement in pain (84.18% Vs 89.17, p>0.05). No statistically significant difference in mean change in NPSI total score from baseline was observed (-37.57 Vs -38.47, p>0.05).\n\nWhen patients were asked about their global impression of improvement using the PGI-I, patients reported much better to very much better improvement with both treatments. The mean of the PGI-I score at Week 7 was 1.7 and 1.6 between test group and treatment group (p>0.05).\n\nWhen investigators were asked about their global impression of improvement using the CGI-I, investigators reported much better to very much better improvement with both the treatments. The mean of CGI-I scores at week 7 was 1.6 in both the treatment groups (p>0.05). Table 3 represents the secondary outcome data.\n\nThe percentage of subjects who experienced TEAEs were comparable between the groups. Most of the TEAES were at least possibly related to study treatment. In the reference group, two patients discontinued the treatment due to TEAES while none in the test group discontinued for this reason. Table 4 presents an overall summary of adverse events for the safety population.\n\nThe major side effects reported in both groups were somnolence, dizziness, and oedema (Table 4). All the reported TEAEs were mild in severity. The incidence of peripheral oedema was higher with the reference treatment as compared to the test treatment (3.11% vs. 0%, p>0.05), although statistically not significant.\n\nNo serious adverse events (SAE) were observed in the study.\n\nIn additional analysis, significantly more moderate to severe adverse events were observed in the reference group as compared to the test group. Furthermore, early onset of somnolence and gastrointestinal AEs were observed with pregabalin monotherapy compared to FDC of low-dose pregabalin and duloxetine and this difference was significant. Similarly, significantly, more patients withdrew from the study due to experiencing AEâs or needing treatment for AEâs in the test group compared to the reference group.\n\n\nDiscussion\n\nNeuropathic pain can be spontaneous or provoked and the patient may complain of paraesthesia, dysesthesia, and loss of normal sensation due to nerve damage.21 The most commonly used option to treat neuropathic pain is pharmacotherapy. However, the complex aetiology of neuropathic pain is the reason for the limited benefit of monotherapy.\n\nThe drawback of monotherapy is its limited efficacy and increased adverse effects with the higher dose. Due to tolerability issues, higher doses needed for maximum therapeutic response are not used. Inadequate partial pain relief with the single agent will lead to impaired physical function, impaired sleep, reduced quality of life and higher economic costs of unrelieved pain.\n\nCombination therapy with two or more drugs must be considered to overcome the challenges of managing uncontrolled neuropathic pain with improved analgesic efficacy and reduced overall side effects especially when synergistic interactions are possible for dose reductions of combined drugs.22 In clinical practice, combination therapy is frequently used.\n\nEvidence supports the combination of gabapentinoids and antidepressants than a high dose of individual drugs from either class for a reasonable treatment in neuropathic pain. Pregabalin, a gabapentinoid and duloxetine, an antidepressant; should be initiated with lower doses and gradually up-titrated based on the treatment response.\n\nPregabalin-duloxetine combination shows additive analgesic actions with a better safety profile compared to high-dose pregabalin monotherapy. In clinical practice, lower doses (once daily or twice daily) of pregabalin (50mg, 75 mg) and duloxetine (20mg, 30 mg) are widely co-prescribed by the neurologist. A combination of significantly lower doses of pregabalin-duloxetine has a greater pain relief than for side effects.23\n\nDanish expert recommendation reported that a combination of gabapentinoid and SNRI is associated with better pain relief and lesser side effects than a single drug therapy.24 An Indian consensus statement suggests that when managing neuropathic pain management with gabapentinoids as first drug, TCAs such as nortriptyline or SNRIs can be considered as add on therapy and vice versa.25 The American Diabetes Asssociation (ADA) position statement recommends combination of two first line agents from class of gabapentinoids, SNRIs, or TCAs in cases wherein monotherapy fails. Furthermore, it also suggests that tramadol/tapentadol can be added if two drug combinations provide inadequate pain relief.26\n\nThe COMBO-DN (Combination vs. Monotherapy of pregabalin and duloxetine in DPN) is one of the largest and well known multinational randomized study conducted in patients with diabetic peripheral neuropathy. It reported outcome of duloxetine and pregabalin monotherapy at high doses versus a combination of these two agents at standard dosing.27 The primary outcome of the study was not achieved as no significant difference was measured between the monotherapy and standard-dose combination therapy. However, secondary outcomes of this trial showed that number of patients who achieved >50% reduction were more in combination treatment compared to high dose monotherapy (52.1% Vs 39.3%, p=0.068) with better tolerability.\n\nThe initial evidence indicates the need of additional large clinical trials in neuropathic pain for identifying optimal combination as initial approach. Therefore, we conducted a phase 3 trial on the fixed-dose combination of lower doses of pregabalin (50mg, 75mg) and duloxetine (20mg, 30mg) in adult patients with moderate to severe neuropathic pain.\n\nOur phase 3 trial result suggests that the combination of a low dose of pregabalin and duloxetine provides similar analgesia and better safety in comparison to a high dose of pregabalin (150mg, 300mg) monotherapy. The subjective assessment done by CGI-I and PGI-I was also found similar in both the groups.\n\nData from the present trial suggest that a combination of low-dose pregabalin and duloxetine could be a combination of choice considering the relatively lesser dose administration for pregabalin without compromising efficacy and safety. Additional evaluation of safety data indicated a clear tolerability advantage of FDC over monotherapy for clinically meaningful adverse events associated with monotherapy.\n\nThe efficacy results of previous studies on the combination of pregabalin and duloxetine indicate that a combination therapy might be a reasonable therapeutic approach rather than up-titrating the dose in patients with inadequate pain control or having tolerability problem with a high doses of single first-line agent. The outcome of the present study supports the outcomes of previous studies.\n\nIn our trial, there were no significant safety concerns observed in both the treatment groups. The most common adverse effects observed were dizziness and somnolence, which are in line with the currently known profile of these drugs.\n\nIn this study, the overall frequency of adverse effects was similar with both treatments, except that patient receiving the pregabalin monotherapy had a higher frequency of peripheral oedema than those receiving the pregabalin-duloxetine combination. Additional analysis also established the safety advantage of FDC in AEs of common concern over monotherapy.\n\nThe strength of our study is that it is a double-blind, double-dummy parallel-group design. The use of lower doses of pregabalin and duloxetine in combination reflects current clinical practice.\n\nThe limitations of our study include small sample size, shorter duration of the study, and the design of the study which compared a fixed dose combination of pregabalin and duloxetine only with a high dose of pregabalin not with duloxetine. Patient related outcomes such as sleep interference and emotional functioning were not assessed in our trial.\n\nNevertheless, the trial results unequivocally shows that a low dose pregabalin-duloxetine combination is non-inferior to high-dose pregabalin monotherapy in management of patients with moderate to severe neuropathic pain.\n\n\nConclusion\n\nIn patients with moderate to severe neuropathic due to different aetiologies, the analgesic effect demonstrated by FDC of pregabalin (low dose), and duloxetine was comparable to pregabalin (high dose) monotherapy. The FDC was safe and well tolerated. This is the first Indian clinical trial in which FDC of pregabalin and duloxetine was evaluated. Future studies with larger sample sizes, long-term duration and heterogenous population are necessary to understand the clinical outcome with FDC of pregabalin and duloxetine.",
"appendix": "Data availability\n\nFigshare: Fixed dose combination of low dose pregabalin and duloxetine, or pregabalin monotherapy for neuropathic pain: A double-blind, randomized, parallel-group study, https://doi.org/10.6084/m9.figshare.21755288.v4. 28\n\nThis project contains the following underlying data:\n\n- Diagnosis of Patients_ Listing.xlsx\n\n- Patient Demographics.xlsx\n\n- Patient Medical History.xlsx\n\nFigshare: CONSORT checklist for âFixed dose combination of low dose pregabalin and duloxetine, or pregabalin monotherapy for neuropathic pain: A double-blind, randomized, parallel-group studyâ, https://doi.org/10.6084/m9.figshare.21755288.v4. 28\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\nThe authors wish to thank the participating investigators and the participants of the study. The writing of this article was supported by a medical writer at Medwiz Healthcare Communications Private Ltd.\n\n\nReferences\n\nNICE: Neuropathic pain in adults: Pharmacological management in non-specialist settings. NICE, Clinical Guideline; 2013. Reference Source\n\nFinnerup NB, Attal N, Haroutounian S, et al.: Pharmacotherapy for neuropathic pain in adults: A systematic review and meta-analysis and updated NeuPSIG recommendations. Lancet Neurol. 2015; 14(2): 162â173. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSumitani M, Sakai T, Matsuda Y, et al.: Executive summary of the clinical guidelines of pharmacologic therapy for neuropathic pain: Second edition by the Japanese Society of Pain Clinicians. J. Anesth. 2018; 32(3): 463â478. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBouhassira D, Lanteri-Minet M, Attal N, et al.: Prevalence of chronic pain with neuropathic characteristics in the general population. Pain. 2008; 136(3): 380â387. Publisher Full Text\n\nDieleman JP, Kerklaan J, Huygen FJ, et al.: Incidence rates and treatment of neuropathic pain conditions in the general population. Pain. 2008; 137(3): 681â688. Publisher Full Text\n\nAttal N, Cruccu G, Baron R, et al.: European Federation of Neurological Societies. EFNS guidelines on the pharmacological treatment of neuropathic pain. Eur. J. Neurol. 2010; 17: 1113â1188. Publisher Full Text\n\nMu A, Weinberg E, Moulin DE, et al.: Pharmacological management of chronic neuropathic pain. Review of the Canadian Pain Society consensus statement. Can. Fam. Physician. 2017; 63: 844â852. PubMed Abstract\n\nDworkin RH, OâConnor AB, Audette J, et al.: Recommendations for the pharmacological management of neuropathic pain: An overview and literature update. Mayo Clin. Proc. 2010; 85(3, Suppl): S3âS14. Publisher Full Text\n\nBates D, Schultheis BC, Hanes MC, et al.: A Comprehensive Algorithm for Management of Neuropathic Pain. Pain Med. 2019 Jun 1; 20(Suppl 1): S2âS12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuo ZD, Chaplan SR, Higuera ES, et al.: Upregulation of dorsal root ganglion (alpha)2(delta) calcium channel subunit and its correlation with allodynia in spinal nerve-injured rats. J. Neurosci. 2001; 21(6): 1868â1875. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoulin DE, Clark AJ, Gilron I, et al.: Pharmacological management of chronic neuropathic pain â consensus statement and guidelines from the Canadian Pain Society. Pain Res. Manag. 2007; 12(1): 13â21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrown TR, Slee A: A randomized placebo-controlled trial of duloxetine for central pain in multiple sclerosis. Int. JMS Care. 2015; 17(2): 83â89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTarride JE, Collet JP, Choiniere M, et al.: The economic burden of neuropathic pain in Canada. J. Med. Econ. 2006; 9(1â4): 55â68. Publisher Full Text\n\nHanna M, OâBrien C, Wilson MC: Prolonged-release oxycodone enhances the effects of existing gabapentin therapy in painful diabetic neuropathy patients. Eur. J. Pain. 2008; 12(6): 804â813. PubMed Abstract | Publisher Full Text\n\nMoore RA, Straube S, Wiffen PJ, et al.: Pregabalin for acute and chronic pain in adults. Cochrane Database Syst. Rev. 2009; CD007076. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPritchett YL, McCarberg BH, Watkin JG, et al.: Duloxetine for the management of diabetic peripheral neuropathic pain: response profile. Pain Med. 2007; 8: 397â409. PubMed Abstract | Publisher Full Text\n\nSultan A, Gaskell H, Derry S, et al.: Duloxetine for painful diabetic neuropathy and fibromyalgia pain: systematic review of randomised trials. BMC Neurol. 2008; 8: 29. Publisher Full Text\n\nFreeman R, Durso-Decruz E, Emir B: Efficacy, safety, and tolerability of pregabalin treatment for painful diabetic peripheral neuropathy: findings from seven randomized, controlled trials across a range of doses. Diabetes Care. 2008; 31: 1448â1454. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDooley DJ, Taylor CP, Donevan S, et al.: Ca2+ channel alpha2delta ligands: novel modulators of neurotransmission. Trends Pharmacol. Sci. 2007; 28: 75â82. PubMed Abstract | Publisher Full Text\n\nIyengar S, Webster AA, Hemrick-Luecke SK, et al.: Efficacy of duloxetine, a potent and balanced serotonin-norepinephrine reuptake inhibitor in persistent pain models in rats. J. Pharmacol. Exp. Ther. 2004; 311: 576â584. PubMed Abstract | Publisher Full Text\n\nSchaefar C, Sadosky A, et al.: Pain severity and the economic burden of neuropathic pain in the United States: BEAT Neuropathic Pain Observational Study. Clinicoecon. Outcomes Res. 2014; 6: 483â496. Publisher Full Text\n\nChaparro LE, Wiffen PJ, Moore RA, et al.: Combination pharmacotherapy for the treatment of neuropathic pain in adults. Cochrane Database Syst. Rev. 2012; 2020. Publisher Full Text\n\nGilron I: Combination of pregabalin with duloxetine for fibromyalgia: a randomized controlled trial. Pain. 2016; 157: 1532â1540. PubMed Abstract | Publisher Full Text\n\nFinnerup NB, Attal N, Haroutounian S, et al.: Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol. 2015; 14(2): 162â173. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaxena AK, Jain P, Dureja GP, et al.: Pharmacological management of neuropathic pain in India: A consensus statement from Indian experts. Indian J. Pain. 2018; 32: 132â144.\n\nPop-Busui R, Boulton AJ, Feldman EL, et al.: Diabetic neuropathy: a position statement by the American Diabetes Association. Diabetes Care. 2017; 40(1): 136â154. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTesfaye S, Wilhelm S, Lledo A, et al.: Duloxetine and pregabalin: high-dose monotherapy or their combination? The âCOMBO-DN studyââa multinational, randomized, double-blind, parallel-group study in patients with diabetic peripheral neuropathic pain. Pain. 2013; 154(12): 2616â2625. PubMed Abstract | Publisher Full Text\n\nKrishnaprasad K: Fixed dose combination of low dose pregabalin and duloxetine, or pregabalin monotherapy for neuropathic pain: A double-blind, randomized, parallel-group study. [Dataset]. figshare. 2022. Publisher Full Text"
}
|
[
{
"id": "236036",
"date": "11 Feb 2024",
"name": "Denis Dupoiron",
"expertise": [
"Reviewer Expertise neuropathic cancer pain"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVery good paper, the topic is attractive, the comparative study is well built, and the methodology does not present significant issue. The outcomes are well presented and relevant. The results validate observations in clinical practice. Moreover, the discussion is helpful and well conducted.\nMy only one comment is about limitations, I do think the number of patients included is low.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "254315",
"date": "13 Apr 2024",
"name": "Laith Thamer Al-Ameri",
"expertise": [
"Reviewer Expertise Neurosurgery",
"Neuroanatomy",
"Clinical Trials",
"sleep disorders",
"neuropathy",
"and Artificial Intelligence in medical fields"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Editor, Authors, and Readers,\nA well-written manuscript and good work, such a trial will add much to what is known and previously written about neuropathic pain. Pregabalin is well known to be equivalent to or superior to many other medications in neuropathic pain management, and additional information is needed about the benefits of and combinations with other medications regarding efficacy and tolerability.\nIt's worth noting that the manuscript demonstrates strong enrolment criteria, a robust methodology, and the use of appropriate statistical methods, which contribute to the overall quality of the study.\nHowever, the conclusion section needs to be rewritten to represent the results and be more informative. The authors have to mention the result regarding the efficacy and tolerability of both groups (e.g., a low-dose pregabalin-duloxetine combination is non-inferior to high-dose pregabalin monotherapy in the management of patients with moderate to severe neuropathic pain), a statement already mentioned at the end of the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "254301",
"date": "16 Apr 2024",
"name": "Nariman Essmat",
"expertise": [
"Reviewer Expertise Pain"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSubject: Review of Manuscript The manuscript titled \"Efficacy and Safety of Fixed-Dose Combination of Pregabalin and Duloxetine for Neuropathic Pain\" provides valuable insights into the efficacy and safety of a fixed-dose combination (FDC) of pregabalin and duloxetine compared to pregabalin monotherapy for the management of neuropathic pain. The manuscript provides a clear overview of the study's objectives, methods, results, discussion, and conclusions. However, the paper needs minor revision before acceptance. My comments have been addressed as follows:\nThe title does not specify the phase of the study. Including information about the study phase (e.g., phase 3) would be better In the abstract: in the method section the authors should clarify the method of pain assessment (numeric pain rating scale (NPRS). In the first paragraph of the introduction section, authors should define neuropathic pain according to IASP. In the Study Design and Setting section, please mention that the study is in phase III Please add more information for the inclusion criteria In the statistical analysis section, please explain how the Non-Inferiority Margin was determined. DPN: give the full name for this abbreviation, no need for an abbreviation as it appears one in the text The study was double-dummy; no data was added for the placebo group; please provide supplementary data on this issue In the Primary outcomes section, what does the PP population refer to? In Table 3, what was the statistical test used for the calculation of the p-value? Itâs better to add a table ligand In the conclusion section, it mentioned (In patients with moderate to severe neuropathic due to different aetiologies), please specify the neuropathic pain types that can be treated with this combination\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-353
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https://f1000research.com/articles/12-352/v1
|
30 Mar 23
|
{
"type": "Study Protocol",
"title": "Assessment of depth of invasion in oral squamous cell carcinoma of the tongue: a study protocol",
"authors": [
"Shelley Rajendra Durge",
"Alka Hande",
"Alka Hande"
],
"abstract": "Background: Oral Squamous Cell Carcinoma (OSCC) is the most common malignancy of oral cavity. It remains a leading cause of mortality and morbidity around the world. Overall OSCC represents more than 550,000 cases throughout the world yearly and the 6th leading cause of death. The mortality and rate of OSCC are higher in developing nations. About 25% patients with OSCC of tongue exist with occult metastasis at initial presentation. The utilization of TNM staging of OSCC to assess the clinical response to treatment and survival result is for many years. Prognosis of malignancy also depends on the histopathological grading of the OSCC. Histopathological assessment by measuring Depth of Invasion (DOI) enables us the insight about the extent of invasion by neoplastic cells. This observation will further be helpful for treatment management protocol particularly in OSCC of tongue. Hence, we aimed to assess the significance of DOI in the prognosis and clinicopathological features and survival of OSCC lesions in the current study. Methods: The surgically operated cases of OSCC of tongue from year 2009 to 2015 will be retrieved from the archival. Measurement of DOI will be carried out using Research microscope. The staging of the patients will be done according to the American Joint Committee of Cancer staging system.\n\nThe present study will find DOI in tongue OSCC and its correlation with the clinicopathological features of OSCC of tongue and three years disease-specific survival.\nConclusion: Measurement of the DOI ought to be included as a part of routine histological assessment. This likewise should be utilized for grading the tumor, which can further help in treatment management protocol.",
"keywords": [
"Keywords: âOral squamous cell carcinomaâ (OSCC)",
"âDepth of Invasion (DOI)â",
"âTumor Thickness (TT)â",
"âDisease-specific survival rateâ"
],
"content": "Introduction\n\nOral Squamous Cell Carcinoma (OSCC) is the most common malignancy of oral cavity. It remains a leading cause of mortality and morbidity around the world. Overall OSCC represents more than 550,000 cases throughout the world yearly and the 6th leading cause of death.1 The mortality and rate of OSCC are higher in developing nations.2 Worldwide statistics for OSCC show these malignancies are predominant wherever the utilization of tobacco and alcohol found to be high.3\n\nOSCC is seen at various sites in the oral cavity e.g. buccal mucosa, labial mucosa, gingivobuccal sulcus, floor of mouth, retromolar trigone and tongue. The OSCC of tongue is associated with higher mortality than OSCC in other sites.4 This could be due to increased risk of cervical nodal metastasis when contrasted with other sub-site which more likely because of rich lymphatics of tongue and floor of the mouth. About 25% patients with OSCC of tongue exist with occult metastasis at initial presentation.1 In spite of recent advances in diagnosis and treatment of oral malignancy in past several years, the long-term prognosis with five-year survival rates with advanced OSCC of the tongue is poor.5\n\nThe utilization of TNM staging of OSCC to assess the clinical response to treatment and survival result is for many years. Prognosis of malignancy also depends on the histopathological grading of the OSCC. Although, the tumor stage has consistently been an important contributor to the prognosis of the malignancy, tumor grade is additionally a significant factor to prognostication and type of management. Subsequently, the prognosis and management of OSCC rely upon stage and grade of the tumor.6\n\nEarlier, staging was elicited by superficial evaluation of tumor extension, however, current American Joint Committee on Cancer (AJCC), Cancer Staging Manual, 8th edition has incorporated depth of invasion (DOI) into T staging, which by definition is the distance from the reconstructed mucosal surface to deepest level of invasion. It has been shown a significant factor in rethinking about the staging, which is evolving in progression dependent on depth of invasion cut off that is 5 mm and 10 mm.1\n\nFurthermore, current research speculates that, DOI with histomorphological parameters should be included as prognosticating factor in OSCC patients.\n\nHistopathological assessment by measuring DOI enables us the insight about the extent of invasion by neoplastic cells. This observation will further be helpful for treatment management protocol particularly in OSCC of tongue.\n\nHence, in the current study, we aimed to assess the significance of DOI in the prognosis and clinicopathological features and survival of OSCC lesions.\n\n\n\n1. To assess the DOI in OSCC of tongue.\n\n2. To evaluate clinicopathological features of OSCC of tongue.\n\n3. To correlate DOI with the clinicopathological features of OSCC of tongue.\n\n4. To correlate DOI and three year survival rate in OSCC of tongue.\n\n\nMethods\n\nThe present study will be carried out at the âDepartment of Oral and Maxillofacial Pathology and Microbiologyâ, âSharad Pawar Dental College and Hospitalâ, âDatta Meghe Institute of Medical Sciencesâ (Deemed to be University), Sawangi (Meghe), Wardha, Maharashtra, India. Surgically operated cases of OSCC of tongue from year 2009 to 2015 in this institute will be retrieved from the archival of the department.\n\nThis study will be a retrospective analysis for which the required protocol was approved by the Institutional Ethical Committee of Datta Meghe Institute of Medical Sciences, deemed to be University (DMIMS (DU)IEC/2020-21/136 dated 05-02-2021). Thirty clinically and histopathologically diagnosed, surgically operated cases of OSCC Tongue will be included in the study. Patients having previous history of oral cancer, recurrence and/or distant disease and pre-operative chemotherapy, radiotherapy, or surgery excluded from the study. Demographic data pertaining to age, gender, detailed history of relevant habit with its dose and duration, site of the lesion of all the study population retrieved. The clinical staging of patients (Tumour Node Metastasis) was done in line with the American Joint Committee of Cancer staging system. Hematoxylin and eosin-stained tissue sections were obtained from archives of department for histopathological analysis. Three oral pathologists performed histopathological examination and gave grading of all oral cancer cases using Broderâs grading system in a blinded manner independently. Extent of lymph node metastasis was evaluated by histopathological examination of lymph nodes from surgically excised specimens. A pilot study was carried out to appraise inter and intra-observer reliability for scoring of the histopathological parameters. Follow-up information for disease free survival of five years was documented in the records.\n\nTo determine the DOI, tissue section stained with H & E was inspected using light microscope (Leica) at 100Ã magnification.\n\nIn order to measure an object under a microscope we will use two types of micrometers.\n\n1. Ocular Micrometer (OM) â A small glass disc in the center of which there is line etched and divided into 100 divisions.\n\n2. Stage Micrometer (SM) â A glass micro slide, in the center of which a one-millimeter line is etched and divided into 100 divisions.\n\nIn calibration, the stage micrometer will bring into focus and move until one of the graduations corresponds exactly with one of the divisions of the eyepiece micrometer. The true distance (A) will be seen on the stage micrometer, which will correspond to the number of divisions (B) of the eyepiece micrometer disc will then read and this true distance will be then divided by the number of divisions of the eyepiece micrometer giving the distance each division.\n\n(Refers to the value of 1 SM division)\n\nThe number of divisions covered by the specimen multiplied by the calibration constant (C) give the diameter of the specimen.\n\nDiameter of the specimen = C Ã Number of divisions covered by the specimen.\n\nIn this study the DOI will be measured using calibrated eyepiece micrometer. Measurement will be obtained from the basement membrane and deepest point of tumor infiltration.\n\nRetrospective cohort study.\n\nAssociation between the components is carried out by chi square test.\n\nThe present study will find DOI in Tongue OSCC and its correlation with the clinicopathological features of OSCC of tongue and three years disease-specific survival.\n\nPublication in Indexed Journal.\n\n\nDiscussion\n\nOne author studied the possibility of cervical lymph nodal metastasis and the recurrence with respect to depth in OSCC of tongue. They evaluated 179 patients and divided them according to 8th edition AJCC. In group A=1-5 mm; B= 6-10 mm; C>10 mm. They found that the risk of local recurrence and metastasis was higher in group C. So, they concluded that depth more than 10 mm is of increased risk for recurrence and metastasis.1\n\nA study reviewed the importance of depth of invasion in prognosis of oral squamous cell carcinoma. They also reviewed its determination in carcinoma. They said that histological parameter such as DOI might be used as prognostic factor in carcinoma. DOI is ruled out by detecting the invasion, which is deepest in the underlying tissue. After being histologically examined the treatment of patient may be changed. So, DOI measurement should be part of routine histological examination.6\n\nAnother study evaluated the clinicopathological factor with the cervical metastasis in oral squamous cell carcinoma of tongue. They reviewed 44 patients of OSCC of tongue, who were treated with only Partial glossectomy. Within five years, cervical metastasis was developed in 21 patients. Some factors were associated with the development of metastasis they are, nuclear polymorphism, and invasive growth, border of tumor, thickness and depth of invasion. They concluded that stage II or I carcinoma having more than 4mm thickness of tumor is at higher risks for metastasis.7\n\nSome author compared the tumor thickness with the depth of invasion for their effect to determine the eighth edition of American Joint Committee on Cancer T category on survival of OSCC. 927 patients of OSCC were included on this cohort study. Result showed six percent patients had diverse AJCC-8 T category if they use thickness rather than depth. fifteen patients were staged (T2) from (T1) and ten patients (T3) from (T2), whereas there was the same stratification of overall survival found for T category based on DOI and TT.8\n\nAnother author evaluated the literature and impact of depth of invasion in OSCC and its role in predicting mystical cervical lymph node metastasis. There was unambiguous management implication in the review which explains role for elective neck dissection based on DOI. There are large number of studies giving new strategies to treat early stage of OSCC. These altering disease-free survival of OSCC.9\n\nThere is a study which characterised same histopathological findings which complicates in the measurement of depth of invasion in squamous cell carcinoma of tongue like lacking of residual biopsy after biopsy, deep positive margins, extratumoral invasion, lymphatic invasion.10 The squamous cell carcinoma of tongue having less than 4 cm and negative cervical lymph node were taken for study. They concluded that measurement of DOI for OSCC of tongue requires re-examination of biopsy in around 20% of cases because of absence of residual carcinoma in specimens of glossectomy.11â13\n\nEthical approval was obtained from the Institutional Ethical Committee of Datta Meghe Institute of Medical Sciences (DMIMS (DU)IEC/2020-21/136 dated 05-02-2021).\n\nOngoing.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nWe acknowledge the support of Dr. Madhuri Gawande, Head, department of Oral Pathology and Microbiology.\n\n\nReferences\n\nFaisal M, Abu Bakar M, Sarwar A, et al.: Depth of invasion (DOI) as a predictor of cervical nodal metastasis and local recurrence in early stage squamous cell carcinoma of oral tongue (ESSCOT). PLoS One. 2018; 13(8): e0202632. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSelvamani M, Yamunadevi A, Basandi PS, et al.: Prevalence of oral squamous cell carcinoma of tongue in and around Davangere, Karnataka, India: A retrospective study over 13 years. J. Pharm. Bioallied Sci. 2015 Aug; 7(Suppl 2): 491â494. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatil TT, Kowtal PK, Nikam A, et al.: Establishment of a tongue squamous cell carcinoma cell line from Indian gutka chewer. Oral Oncol. 2014; 2014: 1â9. Article ID 286013. Publisher Full Text\n\nBroders AC: Squamous cell epithelioma of skin: A study of 256 cases. Ann. Surg. 1921; 73: 141â160. Publisher Full Text\n\nGonzalez M, Tongue Cancer RA: Updated 2020 Sep 15. StatPearls. Treasure Island, (FL): StatPearls Publishing; 2020 Jan\n\nGhazi N, Ghazi A, Shafiee S, et al.: Importance of depth of invasion in patients with oral squamous cell carcinoma: A re- view article. J. Orofac. Sci. 2018; 10: 3â6.\n\nAsakage T, Yokose T, Mukai K, et al.: Tumor thickness predicts cervical metastasis in patients with stage I/II carcinoma of the tongue. Cancer. 1998 Apr 15; 82(8): 1443â1448. PubMed Abstract | Publisher Full Text\n\nDirven R, Ebrahimi A, Moeckelmann N, et al.: Tumor thickness versus depth of invasion â analysis of the 8th edition American Joint Committee on Cancer Staging for oral cancer. Oral Oncol. 2017; 74: 30â33. PubMed Abstract | Publisher Full Text\n\nVarshitha.: Prevalence of oral cancer in India A. J. Pharm. Sci. Res. 2015; 7(10): 845â848.\n\nShariat-Madar B, Liu JC: Role of depth of invasion in evaluation and management of early-stage oral cavity squamous cell carcinoma. Int. J. Head Neck Surg. 2017; 8(2): 84â88. Publisher Full Text\n\nBerdugo J, Thompson LDR, Purgina B, et al.: Measuring depth of invasion in early squamous cell carcinoma of the oral tongue: positive deep margin, extratumoral perineural invasion, and other challenges. Head Neck Pathol. 2019; 13(2): 154â161. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan WJ, Chia CS, Tan HK, et al.: Prognostic significance of invasion depth in oral tongue squamous cell carcinoma. ORL J. Otorhinolaryngol. Relat. Spec. 2012; 74(5): 264â270. PubMed Abstract | Publisher Full Text\n\nGadbail AR, Chaudhary M, Gawande M, et al.: Oral squamous cell carcinoma in the background of oral submucous fibrosis is a distinct clinicopathological entity with better prognosis. J. Oral Pathol. Med. 2017 Jul; 46(6): 448â453. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "272784",
"date": "12 Sep 2024",
"name": "Gerardo Ferrara",
"expertise": [
"Reviewer Expertise Anatomic Pathology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOral Squamous Cell Carcinoma (OSCC) is currently staged with a pT parameter which reflects both the diameter and the depth of invasion (DOI), the latetr being defined as the distance of the deepest point of tumor invasion takenas a 'plumb line' from the basal membrane relative to the the closest intact squamous mucosa. DOI is therefore a well recognized prognostic factor in OSCC. The present study is aimed at validating DOI as a staging parameter by retrospectively collecting surgically operated cases of OSCC of tongue from year 2009 to 2015. In addition, DOI will be correlated with the other clinicopathological features of the collected cases of OSCC Main comment - Since DOI is a well-established prognostic criterion, the study should mainly focus of the association of DOI with other parameter (such as clinical diameter of the tumor, WPOI, perineural and/or lymphovascular invasion, grading. Minor comment - The Authors should clarify whether pT4 cases will be included into the study and, if so, how DOI will be measured in such cases.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-352
|
https://f1000research.com/articles/12-351/v1
|
30 Mar 23
|
{
"type": "Research Article",
"title": "Evaluation of the interaction between ketone bodies and obesity-associated proteins: an in silico approach",
"authors": [
"Omowumi Kayode",
"Deborah Yoko",
"Abolanle A.A. Kayode",
"Deborah Yoko",
"Abolanle A.A. Kayode"
],
"abstract": "Introduction: Obesity is an upsurge in body fat and is associated with a number of cardiovascular and metabolic conditions, including type-2 diabetes, atherosclerosis, dyslipidaemia, hypertension, and several malignancies. The ketogenic diet, which is high in fat and protein and very low in carbohydrates, has become one of the most researched options for weight loss in recent years. It has also recently gained recognition as a metabolic therapy for its efficacious methods in the prevention and treatment of cancer, type 2 diabetes, obesity, and other illnesses. Methods: This study was carried out to investigate the interaction of ketogenic diet end products, the ketone bodies (acetoacetate, acetone and beta-hydroxybutyrate) and standard drugs (orlistat and cetilistat) on selected obesity-related proteins including ghrelin, leptin, fat mass and obesity-associated (FTO) gene protein (PDB id: 3LFM), catalase, superoxide dismutase and 3-hydroxyl-3-methylgluatarate Co-A (HMG CoA) reductase in vivo. Results: In silico docking simulations of the proteins and ligands (standard drugs and ketone bodies) was done using high computing tools and software. The results revealed varied docking scores based on interactions between the proteins and ligands. The standard drugs and ketone bodies exhibited good docking scores for all the proteins docked, although the standard drugs had slightly higher scores in most cases except for FTO, for which the ketone bodies had higher docking scores. This implies that the FTOâketone bodies complex might activate the inhibition of fatty acid synthesis, leading to reduction in stored fat. Conclusions: This study concludes that ketone bodies obtained from ketogenic diets may serve as an adjuvant therapy in the management of obesity with a reduced risk of toxicity compared with conventional therapy.",
"keywords": [
"obesity",
"ketogenic diet",
"ketone bodies",
"proteinâligand biomarkers",
"in silico"
],
"content": "Introduction\n\nObesity has always been a pre-existing problem and has been recognized as a global pandemic in the 21st century (Ryan et al., 2021). It is described as the anomalous, irregular or disproportionate gain of weight as a result of fat buildup in the body, which adversely leads to the risk of several health-related problems such as hypertension, type 2 diabetes, cardiovascular diseases and certain cancer types, amongst others (Aronne, 2002; Natalia et al., 2021).\n\nObesity calculation is based on body mass index â¥30 kg/m2, which is the measurement of fat buildup calculated by dividing body mass (kg) by the square of the body height (m2). In 2016, the World Health Organization (WHO) acknowledged that nearly 2-billion adults worldwide were considered to be overweight and 650 million of that population were found to be obese. The WHO also estimated that 50% of the population found in Europe had a preeminent body weight which could lead to obesity over time (https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight).\n\nFurthermore, in 2002, certain rural areas in Nigeria were recorded to have a high rate of obesity, at 33.7% (Ogah et al., 2013). Obesity involves a complex process, and certain physical and biochemical factors have been found to induce this condition some of which include unhealthy diets and eating habits, high calorie food consumption, and genetic, epigenetic, and ecological factors. All these factors combined together with the lack of physical activity to burn excess body weight then lead to energy disproportion and fat deposition (Williams et al., 2015; Lin et al., 2017). Due to obesity prevalence over time, several solutions and control measures have been brought up to ameliorate as well as control the condition. Some of these measures include physical activity programmes, behavioural lifestyle programmes, pharmacotherapy, diet management, and in severe obese conditions bariatric surgery is recommended (Gonzalez-Muniesa et al., 2017).\n\nCaloric inhibition, a diet management scheme and a nutritional strategy are the commonly used weight loss mechanisms as far as diet management is concerned, and this research focuses on the use of a ketogenic diet, which has been used successfully in the therapeutic treatment of epilepsy, as a control measure for obesity (Anton et al., 2017; UÅamek-Koziol et al., 2019).\n\nA ketogenic diet is made up of a high fat content, very minimal carbohydrate (not exceeding 4 percent) and a sufficient amount of protein. As a result of the proportionate amount of nutrients in the diet composition, there is reduced metabolism of carbohydrate and protein but increased metabolism of fat (Kayode et al., 2020a, 2020b, 2021). Consequent fat breakdown and reduced carbohydrate and protein breakdown then leads to reduced blood glucose levels and the stimulation of ketogenesis in the liver, which produces increased levels of ketone bodies and fatty acids (Kulak and Polotsky, 2013). The resulting ketone bodies are then transported to the bloodâbrain barrier to make available energy for the brain, and increased levels of the ketone bodies lead to increased levels of substrates such as creatine, adenine triphosphate, and phosphocreatine that are essential in the brain (Kayode et al., 2021). This study aims at exploring the efficiency of ketogenic-diet-generated ketone bodies in the prevention and amelioration of obesity using in silico simulations.\n\n\nMethods\n\nThe data retrieval and computation of the entire work design utilised Discovery Studio (DS) version 21.1 (RRID:SCR_015651), Open Babel (RRID:SCR_014920), Python enhanced molecular graphics tool 1.3 (PyMOL 1.3) (RRID:SCR_000305), PyRx (RRID:SCR_018548), Chimera (RRID:SCR_002959), PubChem (RRID:SCR_004284), Protein Data Bank, SWISS-MODEL (RRID:SCR_013032), and Universal Protein resource (UniProt) (RRID:SCR_002380).\n\nThe X-ray crystallographic structures of the human target proteins were downloaded from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) (www.pdb.org) (RRID:SCR_012820) and prepared for molecular docking simulation using DS v. 21.1., and Chimera software. The protein targets not readily available on RCSB PDB were developed via homology modelling using the SWISS-MODEL webserver and further prepared for docking analysis.\n\nDocking analysis was performed according to Sharma et al.âs (2019) protocol. The active binding sites of the protein targets, listed in Table 1, were mapped out using the native ligand interaction and the models without native ligands were determined using the Computed Atlas of Surface Topography of proteins (CASTP) webserver. Molecular docking was performed by using AutoDock Vina (RRID:SCR_011958) software (Trott and Olson, 1995) in PyRx platform (GUI version 0.8).\n\n* No interaction.\n\nDetermination of the structural interactions of the proteinâligand complex result was performed using DS v. 21.1. software (Kayode et al., 2023).\n\n\nResults and discussion\n\nLeptin aids hunger inhibition and energy intake-energy output balance. In the docking analysis results collated from the molecular docking of the protein, leptin was shown to exhibit a positive binding interaction with the ketogenic diet by-products (see Figures 1, 2 and 3 (β-hydroxybutyrate -3.7, acetoacetate -3.3, acetone -2.5)), as well as the standard drugs orlistat, -4.7, and cetilistat, -5 (Figures 4 and 5) used during this research study, although interactions were higher with standard drugs than with the ketone bodies. Therefore, the interaction between the standard drugs and ketone bodies with leptin indicates that greater levels of hunger inhibition, which will culminate in weight reduction, may emanate from standard drug and ketone bodies treatment since leptinâs function of inhibiting hunger and inducing starvation is highly effective in obesity treatment.\n\nStimulation of appetite through the hypothalamic arcuate nucleus, which controls food input and output, is achieved by the ghrelin protein (Kojima and Kangawa, 2005), as shown in the docking analysis results in Figures 6â10. The standard drugs (orlistat, -5.2, and cetilistat, -6.3) and ketone bodies (β-hydroxybutyrate -3.1, acetoacetate -3, acetone -2.3) exhibited positive binding interactions with the ghrelin protein, with the standard drugs having an increased interaction compared with the ketone bodies, which had minimal binding interaction. This connotes that the ghrelin function in the presence of these ligands (standard drugs and ketone bodies) might be inhibited and thus results in the lowering of appetite and thus food consumption, leading to weight loss in individuals.\n\nThe docking analysis of catalase interaction with standard drugs and ketone bodies (Figures 11â15) revealed the catalase exhibited a positive binding interaction with standard drugs at a higher level than ketone bodies, thereby showing that the catalase function of catalysing the disproportionate amounts of hydrogen peroxide synthesised in cells by certain enzymes and oxidases into a single oxygen (O2) molecule and water (H2O) molecule, and maintaining optimal compound levels which induce cell signalling by deactivating hydrogen peroxide are activated by the ketone bodies. This will enhance the anti-oxidative role of the diet and, since oxidative stress has been implicated in obesity progression, an enhanced cell anti-oxidative status by the ketone bodies will lead to the amelioration of obesity (Everse, 2013).\n\nThe docking analysis result of superoxide dismutase (SOD) (Figures 16â20), which is similar to that of catalase, showed positive binding interactions with both standard drugs (orlistat, -5.0, and cetilistat, -5.7) and ketone bodies (β-hydroxybutyrate -3.7, acetoacetate -3.6, and acetone -2.5), hence the SOD function of promoting the defence mechanism of cells against Reactive Oxygen Species (ROS) by catalysing the oxidative deamination of superoxide radicals (O2) into oxygen molecules (O2) and hydroxyl radicals (H2O2) are activated in vivo by the standard drugs and ketone bodies. This will also enhance the reduction of oxidative stress thus promoting amelioration of metabolic syndromes associated with obesity.\n\nThe docking analysis result collated for the FTO interaction with standard drugs (orlistat, 1, and cetilistat, 0.3) and ketone bodies (β-hydroxybutyrate -4.7, acetoacetate -4.5, acetone -3.2), shown in Figures 21â25, showed minimal binding interactions with the standard drugs but showed increased binding interactions with the ketone bodies ligands. This indicates that orlistat and cetilistat in the biological system may have minimal inhibitory action against FTO, while the ketone bodies exhibit greater inhibition of FTO. which can lead to the downregulation of fatty acid synthesis and storage, leading to significant reduction in body fat and obesity.\n\nThe docking analysis for the 3-hydroxyl-3-methylgluatarate Co-A (HMG-CoA) reductase interaction with the standard drugs (orlistat, -4.7, and cetilistat, -4.7) and ketone bodies (β-hydroxybutyrate -3.4, acetoacetate -3.4, acetone -2.5) is shown in Figures 26â30. HMG-CoA exhibited binding interactions with the standard drugs and ketone bodies. The interactions may result in inhibition of HMG-CoA reductase, hence leading to reduction in the levels of mevalonate and cholesterol synthesised in vivo This will consequently result in body fat reduction over a period of time.\n\n\nConclusions\n\nThe ketone bodies exhibited varied positive interactions with the obesity-associated proteins and thus, sequel to further in vivo studies, the ketogenic diet may emerge as a therapeutic remedy for weight maintenance and obesity inhibition, which works via the ketone bodiesâ interaction with obesity-associated proteins besides other mechanisms.",
"appendix": "Data availability\n\nFigshare: Underlying data for âEvaluation of the interaction between ketone bodies and obesity-associated proteins: an in silico approachâ. moleculardockingand2d3dinteractionsresults.zip. https://doi.org/10.6084/m9.figshare.21804411.v1. (Kayode, 2023).\n\nThis project contains the following underlying data:\n\n- LIGAND and PROTEINS for docking 2.docx\n\n- MOLECULAR DOCKING AND 2D-3D INTERACTIONS RESULTS.docx\n\n- 3 HYDROXYL RESULT.csv\n\n- AROMATES RESULT.csv\n\n- CATALASE RESULT.csv\n\n- FMO RESULT.csv\n\n- FOLLICLE RESULT.csv\n\n- GHRELIN RESULT.csv\n\n- INHIBIN A.csv\n\n- INHIBIN B.csv\n\n- LEPTIN RESULT.csv\n\n- LUTEINISING RESULT.csv\n\n- OESTROGEN RESULT.csv\n\n- SUPEROXIDASE DIMUTASE.csv\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgement\n\nThe authors wish to thank the management of Mountain Top University for financial support for the payment of the article processing fee of this publication.\n\n\nReferences\n\nAnton SD, Hida A, Heekin K, et al.: Effects of Popular Diets without Specific Calorie Targets on Weight Loss Outcomes: Systematic Review of Findings from Clinical Trials. Nutrients. 2017; 9(8): 822. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAronne LJ: Classification of obesity and assessment of obesity-related health risks. Obes. Res. 2002; 10(Suppl 2): 105Sâ115S. Publisher Full Text\n\nEverse J: Heme Proteins. Encyclopedia of Biological Chemistry. 2013; pp. 532â538. Publisher Full Text\n\nGonzález-Muniesa P, Mártinez-González MA, Hu FB, et al.: Obesity. Nat. Rev. Dis. Primers. 2017; 3: 17034. Publisher Full Text\n\nKayode OT, Yoko DU, Kayode AA:moleculardockingand2d3dinteractionsresults.zip. Dataset. figshare. 2023. Publisher Full Text\n\nKayode OT, Damilare ER, Afolayan OA, et al.: Ketogenic Diet: A Nutritional Remedy for Some Metabolic Disorders. J Edu, Health Sport. 2020a; 10(8): 180â188. Publisher Full Text\n\nKayode OT, Rotimi DE, Olaolu TD, et al.: Ketogenic diet improves and restores redox status and biochemical indices in monosodium glutamate-induced rat testicular toxicity. Biomed. Pharmacother. 2020b; 127: 110227. PubMed Abstract | Publisher Full Text\n\nKayode OT, Kayode AA, Mgbojikwe I, et al.: Effect of Ketogenic Diet on Monosodium Glutamate-Induced Uterine Fibroids in Female Wistar Rats. J. Babol Univ. Med. Sci. 2021; 23: 1â8.\n\nKojima M, Kangawa K: Ghrelin: structure and function. Physiol. Rev. 2005; 85(2): 495â522. Publisher Full Text\n\nKulak D, Polotsky AJ: Should the ketogenic diet be considered for enhancing fertility? Maturitas. 2013; 74(1): 10â13. Publisher Full Text\n\nLin X, Lim IY, Wu Y, et al.: Developmental pathways to adiposity begin before birth and are influenced by genotype, prenatal environment and epigenome. BMC Med. 2017; 15(1): 50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNatalia D, WiesÅaw W, Mariusz KP, et al.: Recent advances in the application of a ketogenic diet for obesity management. Trends Food Sci. Technol. 2021; 110: 28â38. Publisher Full Text\n\nOgah OS, Madukwe OO, Chukwuonye II, et al.: Prevalence and determinants of hypertension in Abia State Nigeria: results from the Abia State Non-Communicable Diseases and Cardiovascular Risk Factors Survey. Ethn. Dis. 2013; 23(2): 161â167. PubMed Abstract\n\nRyan D, Barquera S, Barata Cavalcanti O, et al.:The Global Pandemic of Overweight and Obesity.Kickbusch I, Ganten D, Moeti M, editors. Handbook of Global Health. Cham:Springer;2021. Publisher Full Text\n\nSharma P, Joshi T, Joshi T, et al.: In silico screening of potential antidiabetic phytochemicals from Phyllanthusemblica against therapeutic targets of type 2 diabetes. J. Ethnopharmacol. 2019; 284: 112268.\n\nTrott O, Olson A: AUTODockVINA Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Protein Eng. 1995; 8: 127â134.\n\nUÅamek-KozioÅ M, Czuczwar SJ, Januszewski S, et al.: Ketogenic Diet and Epilepsy. Nutrients. 2019; 11(10): 2510. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams EP, Mesidor M, Winters K, et al.: Overweight and Obesity: Prevalence, Consequences, and Causes of a Growing Public Health Problem. Curr. Obes. Rep. 2015; 4(3): 363â370. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "187449",
"date": "01 Nov 2023",
"name": "Chirag N. Patel",
"expertise": [
"Reviewer Expertise Bioinformatics"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study focuses on in silico approaches to identify potential inhibitors for ketone bodies and obesity-associated proteins. Although, the study has some positive implications, however, there are several shortfalls and lacunas in the organization and presentation of the data as a whole. Therefore major modifications will be required entirely. Here are some reservations:\n1. It is recommended that the authors should expand the Introduction part with reference to the employed in silico methods in this manuscript. The introduction is not sufficiently highlighted the importance of considering the ketone bodies and obesity-associated protein inhibitors and their application. The authors should have emphasized the structural organization of selected proteins. The rationale of the in silico study is missing in the introduction part. Moreover, the importance of different inhibitors reported so far and such information is missing or the designed study hypothesis is missing in its present form.\n2. How the authors have finalized the proteins? In the abstract part, 3LFM protein was mentioned but the material and methods section lacks this protein details.\n3. Why the authors have chosen the various sequences? Did they perform the comparison?\n\n4. Why the authors did not do the homology modeling in Alphafold? What are the results of the homology modeling?\n\n5. How have the authors finalized the active site?\n6. Ligand preparation has not been written. All the subtopics of the materials and methods section need major corrections. Which basis the drugs were selected for the study?\n7. Did the authors have performed the energy minimization of all structures? If yes, please provide the details.\n8. Protein preparation is missing in the manuscript.\n9. Please provide the scripts used for molecular docking for all complexes.\n10. Please provide the formulas for all the required studies.\n11. The conclusion is too chaotic.\n12. Why have authors not accomplished the MD simulation of the docked complexes?\n13. The docking results were improperly explained and it is not addressed well it requires more accuracy.\n14. The references of all software and algorithm used in the study have to be cited in the text.\n15. Can the author provide the license details of all listed software?\n16. What are the homology modeling results? It must be incorporated.\n17. The results were not discussed properly.\n18. Did the authors have validated the docking study?\n19. This study must be validated by experimental methods.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "217748",
"date": "01 Nov 2023",
"name": "Endre Kristóf",
"expertise": [
"Reviewer Expertise Adipose tissue biology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript contains extensive amount of in silico data simulating how ketones and certain drugs bind to proteins which have critical roles in the regulation of energy metabolism. On one hand, the manuscript presents interesting and clinically important data and it is generally well written. On the other hand, the study lacks functional experiments and therefore the conclusions are sometimes seem to be speculative. Moreover, the presentation of figures should be significantly improved.\nThe followings should be revised before the final publication of the manuscript:\nIt should be explained in the Interaction why the presented proteins of interest were chosen of the in silico analysis and their role in energy metabolism should be described shortly with appropriate references.\n\nI suggest to rationalize the number and restructure the figures by including negative or confirmatory data in a supplementary datasheet.\n\nFigure legends should contain more details: brief description of the experimental process and the data that are shown on each subfigure. The data displayed in the figures cannot be understood without a well written caption.\n\nThe Authors show important amino acid residues probably involved in the binding of the investigated compounds, however, these data are not described in the manuscript text.\n\nThe Authors should clearly state in the Conclusion which of their presented findings are novel.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-351
|
https://f1000research.com/articles/11-170/v1
|
11 Feb 22
|
{
"type": "Research Article",
"title": "Awareness and intention to register halal certification of micro and small-scale food enterprises",
"authors": [
"Hirawati Oemar",
"Endang Prasetyaningsih",
"Siti Zakiah Abu Bakar",
"Djamaludin Djamaludin",
"Anis Septiani",
"Hirawati Oemar",
"Siti Zakiah Abu Bakar",
"Djamaludin Djamaludin",
"Anis Septiani"
],
"abstract": "Background: This paper discusses halal awareness of food micro and small-scale enterprises (food MSEs) in West Java Province, Indonesia. Halal awareness is the first step toward obtaining halal certificates, which confirm that the product is lawful according to Islamic Sharia. Unfortunately, most of the food sold on the market do not have halal certificates due to a lack of halal awareness and intention on the part of the entrepreneurs. Methods: This study aims at measuring the level of halal awareness and the intention of food MSE entrepreneurs to register halal certification. Halal awareness is assumed to be influenced by knowledge of halal and MSEs' entrepreneurial perceptions of the benefits of halal certificates. Furthermore, halal awareness, attitudes, and perceptions of ease of procedures will encourage the intention to register halal certification. An electronic Google Form with a cover letter and a set of questionnaires was distributed to collect data. Structural Equation Modelling â Partial Least Square (SEM-PLS) was chosen to evaluate the adopted theoretical models in the exploratory research. Results: The results show that halal awareness is influenced by knowledge of halal and perceptions of benefits. Moreover, halal awareness influences positively the intention to obtain a halal certificate, but the intention is not significantly affected by attitudes and perceptions of procedures for obtaining halal certification. This shows that halal awareness will increase the intention to register halal certification. However, it does not impact attitudes/actions to register for halal certification due to the misconceptions about the procedures for obtaining halal certificates. Conclusions: Micro and small entrepreneurs in West Java Province, Indonesia have a good level of awareness about halal food. However, their products are not halal-certified due to the perceptions of the procedures for obtaining halal certificates, which are relatively complicated and costly for micro and small-scale businesses.",
"keywords": [
"halal awareness",
"intention",
"halal certification",
"food micro and small enterprise",
"Islam"
],
"content": "Introduction\n\nRecently, the halal industry is a fast-growing market globally with a growth rate of 20% per year due to the average growth of the global Muslim population of 1.8%.1 There are approximately 1.6 billion Muslims in the world's population, who live in over 100 countries, and this figure is expected to rise to 2.2 billion by 2030.2\n\nThe globalization of the halal industry provides an opportunity for local micro and small-scale enterprises (MSEs) to sell halal products. Local MSEs, on the other hand, are hesitant to compete in the global market because they lack halal certificates, which are one of the most important requirements for entering the global market. Halal certifications are also regarded as a quality-control standard among Muslim consumers. Many non-Muslims have no qualms about eating halal food, but they may react negatively if they eat halal food accidentally and feel cheated.3 As a result, MSEs that want to enter the global market must have a halal certificate.\n\nHalal certificate or halal logo is an important consideration when consumers of both Muslim and non-Muslim,4â6 or Muslim gen Z,7 purchase products, especially for products made by non-Muslim producers.8 The halal logo has even been recognized in Japan9 where Muslims are a minority. With the issuance of âUndang-Undang Republik Indonesia No 33 Tahun 2014â (Law of Republic Indonesia No 33/2014 - English)10 regarding halal product guarantees, the halal certification has become a requirement for producers in Indonesia. Halal certification is a symbol of ethical behaviour in the food industry that can help entrepreneurs expand their businesses11 or as a business expansion marketing tool.7\n\nAlthough Islam is the majority religion in Indonesia, most micro, small, and medium enterprises (MSMEs) do not register their products for halal certification. Only 10% of MSMEs have halal certificates, according to the Association of Food and Beverage Entrepreneurs (GAPMMI) in June 2019.12\n\nIn another case, most of the food MSEs in the nearby area of Universitas Islam Bandung (Unisba), which is located in Bandung, the capital of West Java Province, Indonesia, do not have halal certificates, although they serve thousands of Unisba students and employees on a daily basis. They have limited knowledge of halal products and the procedures for obtaining halal certificates, as well as a lack of desire to obtain halal certificates. They are unconcerned about the halal status of the materials or the food they sell.13 These indicate the food MSEs' lack of understanding and awareness of the importance of halal products. Hence, it's critical to assess halal awareness and the intention to obtain halal certificates.\n\nThe Indonesian Ulama Council (LPPOM-MUI) is a government-appointed institution that issues halal certificates. Each product will receive a halal certificate or halal logo, after going through a certification process that starts with the procurement of raw materials, processing into finished products, storing, and delivering finished products. According to our observations, the majority of food MSEs entrepreneurs in West Java Province, Indonesia, are from low- to middle-income families who are not well-educated. The problem is that food MSEs entrepreneurs may be unaware that raw materials are being processed, or that the processing method does not meet halal standards, resulting in a low intention to obtain a halal certificate. Hence, this study aims at measuring the level of halal awareness and the intention of food MSE entrepreneurs in West Java Province, Indonesia to register halal certification.\n\nThis paper is organized as follows; the literature review, the proposed conceptualizing model, the research method, the results, discussion of the results, the conclusions, and data availability.\n\n\nLiterature review\n\nStudies of halal awareness have been carried out in the last decade. Research of Giyanti and Indriastiningsih14 combines awareness and intention as a single variable, claiming that awareness/intention is influenced by knowledge of halal, perceived benefits, and perceived procedures. However, Dinev and Hu,15 Bachok et al.,16 and Rezai and Teng17 show that customer awareness is a strong predictor of their intention to buy or select a product. The influenced factors of halal awareness in this study are derived from Giyanti and Indriastiningsih,14 Dinev and Hu,15 Bachok et al.,16 and Rezai and Teng17 and described in the following explanations.\n\nIn general terms, halal means permitted, authorized, allowed, lawful, legal, or slick according to Islamic Sharia.18 As described in the Law of Republic Indonesia No. 33/201410 Halal products are those that conform to Islamic Sharia (principles). Carrions, blood, pigs, and/or halal animals (e.g., chicken, cow, goat, etc.) slaughtered in a manner inconsistent with Islamic Sharia are all considered non-halal materials. Furthermore, non-halal materials also include intoxicating plants or drinks, material that is harmful to one's health, and microbes contaminated with non-halal materials. Halal encompasses substances (dzatihi), the nature of the substances, processes, processing areas, processing instruments, product storage, product distribution, and serving.19 Based on these explanations, this study defines halal as what is permissible for Muslims to eat, drink, and use under Islamic law.\n\nAwareness is defined as the state of being aware: knowledge and understanding that something is happening.20 According to the definition of halal used in this study and the definition of the word awareness in the dictionary, halal awareness is then conceptualized as a process of being aware of what is allowed for Muslims to eat, drink, and use.\n\nThe level of halal awareness is influenced by religious beliefs, exposures, the role of halal certification through the halal logo/label, and health-related reasons.21 The gender and marital status of SMEs in Thailand have varying levels of awareness to register halal certification. Halal food certificates are required to increase self-confidence, customer trust, and customer satisfaction, although SMEs in Hat Yai, Thailand are dissatisfied with the poor dissemination of halal hub information.22 Halal transportation and halal warehouses also demonstrate SMEs' level of halal awareness in order to maintain the integrity of halal products stored and shipped.23\n\nHalal awareness does not belong solely to halal product manufacturers. Non-halal restaurant owners in Manila, Philippines are generally aware of the 12 halal certification standards (raw materials, tools and equipment, facilities, buildings, exterior areas, location, halal documentation, staff characteristics, staff policies, pest controls, management responsibilities, and waste management), and the majority are âWillingâ to be halal certified.24\n\nAccording to our observations, the majority of micro and small-scale food entrepreneurs in West Java purchase raw materials and process them into finished products without storing and shipping. As a result, the scope of halal awareness in this study is limited to the awareness of using halal materials and processing in a halal manner.\n\nIn the Merriam-Webster dictionary, knowledge is a fact of knowing something with familiarity gained through experience.25 According to the definition of halal used in this study and the definition of the word knowledge in the dictionary, knowledge of halal is then conceptualized as the process of gaining an understanding of what is permissible for Muslims to eat, drink, and use.\n\nThe understanding of halal considers knowledge of the laws relating to halal products described in the Quran and Hadith. In general, all foods are permitted except for those derived from prohibited animals such as pigs, dogs, and carrion, as well as foods and beverages containing alcohol and other toxic or dangerous substances. Slaughter must be carried out in accordance with Sharia, with the intention of doing so in the name of God.2 Allah says in the Quran Surah (chapter) 2 (Al Baqarah) ayah (verse) 173 as follows26:\n\nHe has only forbidden to you dead animals, blood, the flesh of swine, and that which has been dedicated to other than Allah. But whoever is compelled (by necessity), without (willful) disobedience nor transgressing (the limits) then there is no sin on him. Indeed, Allah is Oft-Forgiving, and Most Merciful.\n\nThese halal foods are revealed in Law of Republic Indonesia No. 33/2014 concerning halal product guarantees Based on the previously defined scope of halal awareness, knowledge of halal in this study only includes knowledge about halal/non-halal (haram) materials according to the Quran and Hadith, as well as knowledge about the separation of equipment used to process halal/non-halal (haram) materials.\n\nMerriam-Webster defines benefit as something that produces good effects or that promotes well-being: advantage.27 In this study, benefits are conceptualized as the food entrepreneur's perception of the effect to be gained by producing halal food or having halal certificates and labels. The benefits of producing halal food include increasing market share and competitiveness,28 business growth,11 or business development.7\n\nThe procedure is defined as a set of steps that must be completed in a specific order.29 In this study, the procedure is conceptualized as the food entrepreneur's perception of the steps that must be taken to obtain a halal certificate and label. Standards for gaining halal certification in Indonesia are explained in Law of Republic Indonesia No. 33/2014 concerning the guarantees of the halal products,10 whereas in the Philippines it is broken down into 12 standards.24 In this study, food entrepreneurs' perceptions of the procedure for obtaining halal certificates in Indonesia involve the availability of information about the certification process, as well as the fact that the certification process is simple, inexpensive, and quick.\n\nIn the Merriam-Webster dictionary, attitude is defined as a way of thinking that influences one's behaviour,30 while the intention is the thing that you plan to do or achieve: an aim or purpose.31 In this study, attitude is conceptualized as thinking about producing a halal product, while the intention is conceptualized as an act of registering the product to obtain a halal certificate.\n\nAccording to the Planned Behavior Theory, the intention is determined by three independent factors, i.e., attitude toward behaviour, subjective norm, and perceived behavioural control. Supposing that there is a positive attitude that is supported by people around (as a subjective norm) and there is a perception of ease to perform the behaviour under consideration (as a behavioural control), then an individual's intention to behave will be higher.32\n\nFrom an Islamic perspective, every Muslim must have an attitude to like and want to do a good job. In a broader context, attitude means to do good deeds due to Allah (God) loves those who do good as Allah (God) commands in Quran surah (chapter) Al-Baqarah ayah (verse) 19526:\n\nAnd spend in the way of Allah and let not your own hands throw yourselves into destruction. And do good; indeed, Allah loves the good-doers.\n\nAttitude is also associated with two conditions i.e. good (âmahmudahâ) and bad (âmazmumahâ).33\n\nSeveral previous studies show that there is a positive relationship between attitude and intention to buy or choose a product. Consumers' attitudes and perceived prices are the most significant factors influencing consumers' purchase intentions for private labeled food products.34 Attitude is also one of the factors that influence the intention to buy branded content on Webisodes, a branded content strategy that provides hidden communication.35 A positive relationship between attitude and purchase intention toward organic foods has been shown by Yang, et al.36\n\nThis study restricts food entrepreneurs' attitudes including a focus on halal issues, a guarantee of selling halal products, halal product attention, and checking to use halal raw materials. Meanwhile, food entrepreneurs' intentions are limited to being responsive to the certification process, attempting to meet halal quality standards, implementing a halal assurance system, and registering halal certification as soon as possible.\n\n\nConceptualizing halal awareness\n\nMotivation to obtain halal-certified is significantly influenced by religious understanding and motivation to gain profit.19 The awareness/intention to get halal certificates is significantly influenced by a motivation to get profit because SME entrepreneurs generally agreed that Halal Food Certification provided benefits.14 Meanwhile, the procedure for obtaining halal certificates is relatively complex, thereby reducing the intention of SMEs to register halal certification. Meanwhile, Lee and Shin37 found that consumers' awareness of Corporate Social Responsibility (CSR) activities and purchase intentions are positively related. According to the Planned Behavior Theory, the intention is influenced by attitude, while the ease of procedure to obtain halal certificates will affect the intention to register their products to get halal certificates.\n\nReferring to Giyanti and Indriastiningsih,14 Waluyo,19 and Lee and Shin,37 and the Planned Behavior Theory,32 this study identifies that the variables are knowledge of halal (KH), perception of benefits (PB), perception of procedures (PP), halal awareness (HA), attitude to produce halal product (AHC) and intention to register a halal certificate. Measurement items of each variable are then compiled from the previous studies.10,14,19,21â23,28\n\nReferring to Waluyo19 and Giyanti and Indriastiningsih,14 this study considers that halal awareness is influenced by knowledge of halal and MSEs perception of benefit on the halal certificate. Referring to the Planned Behavior Theory, the halal awareness and attitude (as a positive attitude) and MSEs' perceptions of ease of procedures (as a behavioural control) will encourage the intention for registering halal certification. The relationship of these variables represents the conceptual model of halal awareness and intention to halal certification (see Figure 1).\n\nAs can be seen in Figure 1, halal awareness and intention to register halal certificates involve variables of knowledge of halal, perceptions of benefits, perceptions of procedures, and attitudes. Halal awareness is an independent variable, while knowledge of halal and perceptions of benefits are dependent variables. Furthermore, halal awareness, perception of procedures, and attitude to register halal certification are dependent variables, while the intention to register halal certification is an independent variable.\n\nAs can be seen in the conceptual model (Figure 1), the level of halal awareness is influenced by the knowledge of halal which may include MSEs' understanding of the types of non-halal food as mentioned in Law of Republic Indonesia No. 33/2014.10 Therefore, we hypothesize that:\n\nH1: The knowledge of halal/non-halal levels (KH) positively affects Halal Awareness (HA) of food MSEs.\n\nHalal certificates and halal logos are perceived to have benefits in increasing consumer confidence,22 to use as a promotional tool,14 and compete with other producers.22 Hence, halal certificates are expected to improve the MSEs performance. Based on this point of view, we hypothesize that:\n\nH2: Perception of benefits (PB) positively affects Halal Awareness (HA).\n\nHalal awareness is measured by awareness of the importance of using halal materials in producing halal products,24 and perceiving the benefits to be gained despite the process is very strict.22\n\nConsumer awareness of the green concept is a strong predictor of their intention to consume green foods,17 while customer awareness towards the halal logo fosters purchase intention.16 Referring to Ambali and Akbar38 and Ngah, et al.23 the intention considers regulating actions and a sense of responsibility to gain a halal certificate. In this study, halal awareness is expected to influence on intention to register halal certification. Hence, we hypothesize that:\n\nH3: Halal Awareness (HA) positively affects Intention to register Halal Certification (IHC).\n\nMSE entrepreneurs perceive that the procedure to achieve halal certification is complex due to the lack of information from respondents regarding halal certification procedures.14 This will negatively influence the intention of producers to register halal certification. In the light of this, we hypothesize that:\n\nH4: The MSEs' perceptions of ease of the procedures (PP) positively affect the Intention to register halal certification (IHC).\n\nA positive relationship between attitude and intention has been shown by Jaafar, et al.,34 Yang, et al.,36 and Rezai, et al.17 These show that attitude influences intention. Hence, we hypothesize that:\n\nH5: Attitude to Halal Certification (AHC) positively affects the Intention to register halal certification (IHC).\n\n\nMethods\n\nThis study adopts a quantitative method to evaluate the hypothesis, i.e., analyze the data using descriptive statistics. This study has followed the STROBE guidelines/checklist for cross-sectional research.\n\nInitially, 680 micro and small-scale food and beverage entrepreneurs in the West Java MSME community were targeted. However, based on our observations, medium-scale enterprises differ from micro and small enterprises in terms of the entrepreneurs' education and economic status. Medium-scale entrepreneurs have a better education and economy than MSEs. This has an impact on knowledge and understanding of the significance of halal-certified products. Because micro and small-scale entrepreneurs are more profit-driven, regardless of halal or haram, many products on the market are uncertified. Hence, this research focuses on MSEs. Furthermore, we separate the food and beverage enterprises, due to the different halal requirements for raw materials. Finally, the unit of analysis for this research is food MSEs who are members of the MSME group rather than food and beverage MSME.\n\nThe respondent criteria are 1) micro and small-scale food entrepreneurs with an annual sales turnover are less than 2,500 Rupiahs and net assets are less than 500 million Rupiahs (see Table 1); 2) have an ongoing business; 3) do not have a halal certificate. A Google Form with a cover letter and a set of questionnaires were sent out to potential respondents. This method was chosen because of the COVID-19 pandemic outbreak. In addition, the designed questionnaires could be collected without conducting direct visits to the respondents. The respondents could not participate in the survey unless they gave their written consent. Data from food entrepreneurs were collected from March to May 2020.\n\nWe received written permission from the Chairman of the West Java MSME associations to contact these food entrepreneurs in West Java for data collection. To guarantee that there is no conflict of interest in this study, survey responses are kept anonymous.\n\nData were collected by sending electronically to the respondent standard questionnaires modified from previous studies. The questionnaires were re-translated from English to Indonesian, except for those referring to Waluyo19 due to already being in Indonesian. A copy of the distributed questionnaires can be found in Extended Data.39\n\nThe questionnaire was pretested with a small sample of food MSE entrepreneurs before being distributed to the actual respondents. Based on pretest feedback, the wording of some items was refined and modified to guarantee that the validity and reliability of each variable meet the required standard. The question items were scored on a Likert scale of 1 (strongly disagree) to 5 (strongly agree). The follow-up of this plan is described later in the goodness of measurements instruments session.\n\nThe measurement items for the knowledge halal variable were adapted from Giyanti and Indriastiningsih14 and Waluyo.19 The survey section includes four items, such as knowledge of; slaughtering methods (KH1), haram products (KH2 and KH3), and processing equipment (KH4).\n\nThe measurement items for the perceptions of benefits variable were adapted from Giyanti and Indriastiningsih14 and Abdul.22 These items asked about respondents' perceptions of the benefits they would get if they had a halal certificate, such as a promotional tool (PB1), more convincing consumers to buy (PB2), improving business performance (PB3), and more competitive (PB4).\n\nThe measurement items for the perceptions of the procedure variable were adapted from Giyanti and Indriastiningsih14 and Ambali and Bakar.21 This item asked about the respondent's perception of the procedure for obtaining halal certificates in Indonesia, such as the existence of information about the certification process (PP1), the certification process is easy (PP2), cheap (PP3), and fast (PP4).\n\nThe measurement items for the halal awareness variable were adapted from Abdul22 and Law of Republic Indonesia No 33/2014.10 This part of the survey asked about the awareness of respondents about the importance of producing halal food (HA1), using halal raw materials (HA2), the importance of having a halal certificate (HA3), and the rigorous of the halal certification process (HA4).\n\nThe attitude variable measurement items to produce halal products were adapted from Ambali and Bakar21 and Menteri Hukum dan HAM.10 These items include respondents' attitudes to always pay attention to halal issues (AHC1), ensure consumers buy halal products (AHC2), pay attention to halal products (AHC3), and use halal materials (AHC4).\n\nThe measurement items for the variable of intention to register halal certification were adapted from Ngah, et al.,23 Abdul, et al.,28 and Law of Republic Indonesia No 33/2014.10 These items include respondents' intention to be responsive to the certification process (IHC1), strive to meet halal quality standards (IHC2), immediately implement halal assurance system (IHC3), and immediately register halal certification (IHC4). The variables, indicators, and measurement items are described in Table 2.\n\nWe analyzed the chi-square of early and late respondents' responses to see if there was any potential for non-response bias (the first and last 20 percent of responses received). The findings show that there is no significant variation in responses on key metrics between early and late respondents.\n\nData are analyzed with descriptive statistics to provide a description of the respondents' profile, the results of the assessment of the level of knowledge of halal, perceptions of benefits, halal awareness, perceptions of procedure, attitude, and intention to register halal certificates. The research analysis is intended to assess the model and to objectively describe the hypotheses.\n\nThe adopted theoretical models are evaluated using Structural Equation Model-Partial Least Square (SEM-PLS) method because this study is exploratory research to predict certain constructs by focusing on explaining the variance in the dependent variables when examining the model.41 Free Smart-PLS software 3.3.2. is chosen as data processing.\n\nIn this study, both validity and reliability tests are carried out to measure the goodness of the shared questionnaires. Validity is a test of how well the developed instrument measures the particular construct being measured, while reliability is a test of how the developed instrument consistently measures the construct being measured.42\n\nInitially, the questionnaires had been planned to be pretested in a small sample of food MSEs by distributing it directly beginning in March 2020. However, we were unable to meet with the entrepreneurs due to the social distancing caused by the COVID-19 outbreak. Finally, we decided to distribute the electronic questionnaire Google Forms in May 2020. As a result, the data collected reached 100 respondents in just a matter of days. Due to the limitations of the software features used, the distribution of the questionnaire was halted. As a result, all collected data was subjected to validity and reliability tests.\n\nIn this study, measuring validity uses construct, convergent and discriminant validity to check how well the questionnaire measures the constructs that fit with the adopted theory.\n\n1.âConstruct validity\n\nThe construct validity is assessed by looking at loading and cross-loading to identify problem items if there are any. The validity test using cross-loading is patterned that the main loading factor originating from its construct is greater than the correlation value built from these variables on other constructs.43 Table 3 presents an evaluation of validity based on the value of the main loading factor to the value of cross-loading factors with other constructs. As shown in Table 3, the value of the main loading factor of each construct is higher than the value of the loading factor outside of the main loading factor, so it can be concluded that all constructs are declared valid. For example, the loading factor of AHC1 is higher than the loading factor of HA, IHC, KH, PB, and PP.\n\n2.âConvergent validity\n\nConvergence validity is evaluated by comparing loading factor with both Composite Reliability (CR) and Average Variance Extracted (AVE).41,43 The acceptance level for loading factors is more than 0.5, while the acceptance level for CR is more than 0.70, and the AVE value of 0.50 or higher. Table 4 shows that all itemsâ loadings exceed 0,5, while all CR values are higher than 0.7. In this study, the AVEs for the indicators are within the range of 0.736 and 0.854 respectively (Table 4). Hence, the questionnaire fulfills the requirements for convergent validity. Based on Table 4 it can be concluded that all constructs have p-values smaller than 5% as suggested by Budhiasa,43 so it can be stated that all constructs are valid.\n\n3.âDiscriminant validity\n\nThe square root of the AVE of each construct should be greater than its highest correlation with any other construct, according to the Fornell-Larcker criterion.41 Furthermore, the outer loadings of an indicator on a construct should be greater than all of its cross-loadings with other constructs. Table 5 shows that the average variance extracted by the indicators measuring that construct is less than the squared correlations for that construct. To put it another way, the measurement model represents adequate convergent and discriminant validity.\n\nThe Reliability test is evaluated by the Alpha Cronbach value, the most widely used test. The Alpha Cronbach value higher than 0.6 is acceptable in exploratory research.41 As shown in Table 6, the Alpha Cronbachâs are within the range of 0.878 and 0.943, hence it confirms the reliability of the instrument.\n\nThe Ethical Licensing Committee of the Islamic University of Bandung approved this study by Protocol number: 495/B.04/Bak-k/XII/2019. We had provided all respondents with a consent statement after consultation. The written consent to participate from the Chairman of the West Java MSME Community was gained according to document number: 015/SKIP/IV/2020. In the questionnaire, there is a statement that by filling out the questionnaire the respondents gave their consent to participate. Respondents gave their consent to take part when they filled out the questionnaire. Respondents had given their consent truly and without coercion. Furthermore, to protect respondents' rights and privacy, all forms of data obtained will be kept confidential.\n\n\nResults\n\nBarclay, Higgins, and Thompson (1995) in Hair et al.41 explain the Ten Times Rule in determining the number of PLS-SEM samples, which states that the sample size must be greater than (1) ten times the greatest number of formative indicators used to measure a single construct, or (2) ten times the greatest number of structural paths directed at a specific construct in the structural model. In other words, the minimum sample size is equal to 10 times the maximum number of arrows in the PLS path model pointing to the latent variable.41\n\nIn this study, the IHC variable is the latent variable with the maximum number of arrows, i.e., 3 (see Figure 1). As a result of the Ten Times Rule, 3.10 = 30 represents the bare minimum of observations required to estimate the PLS path model depicted in Figure 1. In terms of Cohen's (1992) recommendation for multiple OLS regression analysis, or running a power analysis using the G*Power program, as cited in Hair, et al.,41 33 observations are required to detect an R2 value of about 0.25, assuming a statistical power of 80% and significance level of 5%.\n\nThe questionnaires were distributed electronically to the West Java MSME community. The questionnaires were returned by 376 people. The returned questionnaires were then sorted using predetermined criteria, yielding 137 questionnaires that met the criteria. However, due to the limitations of the software features used, this study only processed 100 questionnaires at random.\n\nTable 7 displays the percentage of respondents for each indicator. As shown in Table 7, 98% of respondents have Islam as their religion (Muslim), 71% are female, and 88% are 26 years old or older. In terms of business size, 89% of respondents are micro-scale entrepreneurs.\n\nTable 8 shows the descriptive statistics for each measurement indicator. The knowledge of halal (KH) has a high average perception value.\n\nFigure 2 shows the path analysis result of halal awareness and the intention to register halal certification using free Smart PLS 3.3.2. software, while Table 9 represents the hypothesis testing results of each path.\n\nIn this section, the path analysis is addressed to ascertain the hypotheses put forward. As can be seen in Figure 2, the R2 value of 0.797 for IHC indicates that 79.7% of the variance in IHC can be explained by HA, PP, AHC. In addition, Figure 2 also shows that HA, PP, and AHC are positively related to IHC among MSEs entrepreneurs with β = 0.699, 0.042, and 0.2, respectively. According to the t-value of the path coefficients, HA has a significant impact on IHC, while AHC and PP do not have a significant impact on IHC.\n\nBesides, the R2 value of 0.826 for HA means that 82,6% of the variance in HA is influenced by KH and PB, with values β = 0,292 and 0,683 respectively. Hence, KH and PB have a strong and significant impact on the awareness of MSE entrepreneurs about the importance of halal products. According to Hair, et al.41 R2 values of 0.75, 0.50, or 0.25 for the endogenous construct, respectively, can be described as substantial, moderate, or weak. The R2 value of an endogenous latent variable (i.e., halal awareness) described by the two predictive constructs in this study is 82.6% (see Figure 2), which is substantial. Hence, KH and PB are genuine predictors of MSEs awareness to produce a halal product. The same goes for the R2 value of the endogenous latent variable (i.e., Intention to register halal certification) is 79,7% which is substantial. However, the genuine predictor of MSEs Intention to register halal certification is only HA.\n\n\nDiscussion\n\nAs can be seen in Table 8, the findings of the descriptive statistics indicate that most of the respondents are highly aware to have a halal certificate because they have a high level of knowledge of halal and generally agree that Halal Food Certification provides benefits. These findings have confirmed the findings of Waluyo,19 that the religious knowledge and motivation to benefit have a significant impact on the awareness of food producers to certify their product. The finding that knowledge of halal has a significant impact on knowledge of halal is different from the finding of Giyanti and Indriastiningsih14\n\nAs shown in Figure 2, awareness of halal certification and intention to register halal certification have a correlation coefficient of 0.699. It means that the awareness of halal certification is a strong indicator of the intention to register halal certification. It aligns with Rezai, et al.17 and Bachok, et al.16 The descriptive statistics also show that most of the respondents have a high intention to register halal certification with a total mean of 4.625 on a 5 scale (Table 8).\n\nThis study finds that attitudes to produce halal products and perceptions of halal certification procedures have a positive correlation with intentions, but both do not significantly affect intentions. According to the Planned Behavior Theory, when there is support and a sense of ease that there are no barriers to behaviour, intention to behave will increase.44 This study shows that intention to register a halal certificate is not supported by the attitude because West Java food MSEs perceive the procedures to obtain halal certification to be complex.\n\nThe findings also show that MSEs are aware of the benefits of the halal certificate. Based on our observation, we find that they do not require a halal certificate because they have satisfied with the sales/performance that has been achieved.\n\nAnother factor that hinders the desire to obtain a halal certificate is the lack of consumer pressure. Furthermore, MSEs are unaware of the risks of violating the halal product guarantee law if they do not have a halal certificate. Food MSEs' entrepreneurs' lack of understanding of the benefits and risks of having a halal certificate is alleged to lead to that attitude having little bearing on their desire to register for halal certification\n\nThese are the study's main findings. We observe that the local community culture and mindset of micro and small-scale food entrepreneurs appear to be driving this lack of intention to obtain a halal certificate. In general, West Java food MSEs are low- to middle-income communities with limited educational opportunities, so they rarely have broad perspectives or are willing to progress and develop. It is already a good thing that they can sell every day without having to develop their business. They are unaware that halal certificates will increase consumer trust and help food businesses compete more effectively. Halal certificates will therefore increase sales and revenue. Furthermore, by selling halal products, they have aided people who follow the Islamic faith to practice the Quran surah (chapter) 2:(Al Baqarah, ayah (verse) 16826:\n\nO mankind! Eat from whatever is on the earth - lawful and good and do not follow the footsteps of Shaitaan devil. Indeed, he is your clear enemy.\n\nMSEs' perceptions of complicated and costly certification procedures for their scale of operation also hampered their desire to register for halal certification. This finding supports Giyanti and Indriastiningsih,14 whose study found that MSEs' food awareness/intention is hampered by a lack of socialization and the complexity of the procedure for handling halal certification. As a result, socialization and training on halal certification procedures will raise the level of intention.\n\nSocialization and training on halal awareness, halal guarantee system, and halal certification for street vendors around Universitas Islam Bandung (Unisba) which is located at Bandung, the Capital of West Java Province Indonesia have been conducted by Oemar et al.13 The trainees gain a better understanding and awareness of halal food as a result of the training. Consequently, all trainees intend to obtain a Halal Certificate. However, there are several barriers to obtaining halal certificates, including costs (68%), time (24%), and procedures (28%).\n\nThis research could aid efforts to increase food entrepreneurs' desire to obtain halal certifications. Micro and small businesses must be educated about the benefits of obtaining halal certificates and the ease with which they can do so. Micro and small food entrepreneurs' halal awareness should be reinforced by the availability of a readily accessible halal information centre and innovative ecosystems.\n\n\nConclusions\n\nIt can be concluded that micro and small entrepreneurs in West Java Province, Indonesia have a good level of awareness about halal food even though they do not have a halal certificate. They pay attention to the halalness of the material used and of its processing. However, the perceptions of the procedures to obtain halal certificates which are relatively complicated and expensive for micro and small-scale businesses discourage the MSEs to register halal certification.\n\nThe hypothesis test shows that knowledge and perceptions of benefit have positive and significant correlations to halal awareness. In addition, halal awareness, attitude, and perception of the procedure have a positive influence on the intention to register halal certification. However, attitude and perception of procedure do not have a significant impact, while halal awareness has a significant effect on intention. This shows that halal awareness of micro and small-scale food entrepreneurs can increase intention to register their products to be halal certified. However, the reality shows that many products sold in the market do not have halal certificates. It indicates that the halal awareness of micro and small-scale entrepreneurs does not have an impact on real actions to register halal certification. They will take action when they gain real benefit/profit.\n\nFinally, we recognize that this study has some limitations, including 1) The study focuses on micro and small food businesses. Perhaps in the future, research can be done for medium-scale food businesses that have a unique character due to the entrepreneur's higher level of education. 2) The research was conducted in West Java, which has a distinct culture and mindset. In the future, perhaps research can be conducted in other Indonesian provinces with different characteristics. 3) The research was conducted for the food industry. Perhaps, study on other products will be possible in the future. 4) There are six variables in this study. Other estimated variables may be added in the future.\n\nFurther research may be undertaken to measure the level of halal awareness and intention to obtain the halal certificate for medium-scale entrepreneurs with other kinds of products in the other provinces and consider other related variables.\n\n\nData availability\n\nFigshare: Dataset of Questionnaire Result from the respondents of Awareness to Register Halal Certification https://doi.org/10.6084/m9.figshare.17078381.v1.45\n\nThis project includes the following underlying data:\n\n- Questionnaire results from 100 West Java micro and small food entrepreneurs.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: List of questions of descriptions of the questionnaire - Awareness to Register Halal Certification. https://doi.org/10.6084/m9.figshare.17078336.v1.39\n\nThis project includes the following extended data:\n\n- A copy of the questionnaire\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: The Respondent characteristics of Awareness to Register Halal Certification. https://doi.org/10.6084/m9.figshare.17078354.v1.46\n\nThis project includes the following extended data:\n\n- Profile of respondents\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgments\n\nThe authors would like to thank the Rector of Universitas Islam Bandung for his support and for creating a conducive research environment. Furthermore, we would like to thank the members of the Industrial Engineering Department of Universitas Islam Bandung for their support in this research. The author also thanks the Chairman of the Regional Management Board for the West Java MSME community for allowing the distribution of questionnaires, as well as the West Java SMEs Community members who participated in this study. Also, a heartfelt thank you to the Research Synergy Foundation for the recommendations.\n\n\nReferences\n\nDar H, Azmi N, Rahman R, et al., editors. The Global Halal Industry: an Overview Chapter 13. Global Islamic Finance Report. Edbiz Consulting Ltd.; 2013. Reference SourceReference Source\n\nInternational Trade Center: 2015; From niche to mainstream Halal Goes Global. 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Publisher Full Text\n\nKrishnan S, Omar CMC, Zahran I, et al.: The Awareness of Gen Zâs toward Halal Food Industry. Management. 2017; 7: 44â47.\n\nYunus NSNM, Rashid WEW, Ariffin NM, et al.: Muslimâs Purchase Intention towards Non-Muslimâs Halal Packaged Food Manufacturer. Procedia. Soc. Behav. Sci. 2014; 130: 145â154. Publisher Full Text\n\nKawata Y, Htay SNN, Syed AS: Non-Muslimsâ acceptance of imported products with halal logo: a case study of Malaysia and Japan. J. Islam Mark. Artic. 2013; 4: 2013â2014.\n\nMenteri Hukum dan HAM (Minister of Law and Human Rights): Undang-Undang Republik Indonesia No 33 Tahun 2014 (Law of Republic Indonesia Number 33/2014).2014; 1â40.\n\nYusuf AH, Shukor SA, Bustamam USA: Halal Certification vs Business Growth of Food Industry in Malaysia. J. Econ. Bus. Manag. 2016; 4: 247â251. Publisher Full Text\n\nWahyudi E: Baru 10 Persen UMKM yang Kantungi Sertifikat Halal.2019. Reference Source\n\nOemar H, Achiraeniwati E, Rejeki YS, et al.: Improving the Awareness of Providing Halal Food Among Street Vendors. Adv. Soc. Sci. Educ. Humanit. Res. 2020; 409: 17â20.\n\nGiyanti I, Indriastiningsih E: Effect of SME Food Entrepreneurs Knowledge on Halal Certification for Certified Awareness Using Partial Least Square. J. Tek. Ind. 2019; 20: 36.\n\nDinev T, Hu Q: The centrality of awareness in the formation of user behavioral intention toward protective information technologies. J. Assoc. Inf. Syst. 2007; 8: 386â408. Publisher Full Text\n\nBachok S, Jamalluddin N, Arsat A, et al.: Customer Awareness and Behavior Intention Towards the Use of Halal Logo on Restaurants. The Winners. 2011; 12: 196. Publisher Full Text\n\nRezai G, Teng PKZM, Shamsudin MN: Consumersâ awareness and consumption intention towards green foods. Afr. J. Bus. Manag. 2012; 6: 4496â4503.\n\nWahab AR: Guidelines for the preparation of halal food and goods for the Muslim consumers. AmalMerge Sdn Bhd. 2004: 1â12.\n\nWaluyo W: Pengaruh Pemahaman Agama, Motifasi Mendapatkan Profit Dan Tingkat Pendidikaan Terhadap Kesadaran Sertifikasi Halal Bagi Produsen Makanan Di Kabupaten Sleman Dan Bantul. Inf. Dent. 2013; 7: 75.\n\nMerriam-Webster: Halal. Merriam-Webster.com dictionary. Reference Source\n\nAmbali AR, Bakar AN: Peopleâs Awareness on Halal Foods and Products: Potential Issues for Policy-makers. Procedia. Soc. Behav. Sci. 2014; 121: 3â25. Publisher Full Text\n\nAbdul M: Perceptions on Halal food certification in Hat Yai, Thailand. Int. J. Econ. Manag. 2014; 8: 178â194.\n\nNgah AH, Zainuddin Y, Thurasamy R: Adoption of Halal Supply Chain among Malaysian Halal Manufacturers: An Exploratory Study. Procedia. Soc. Behav. Sci. 2014; 129: 388â395. Publisher Full Text\n\nLiba RT, Matsuzawa MP, Coronel JV, et al.: Awareness and willingness on halal certification of non-halal restaurant owners in Manila. TEAM J. Hosp. Tour. 2018; 15: 51â63.\n\nMerriam-Webster; Knowledge. Merriam-Webster.com dictionary. (accessed 19 July 2020). Reference Source\n\nShaikh S, Khatri K, editors. The Glorious Quran Word-for-Word Translation to facilitate learning of Quranic Arabic. Volume 1, juz 1-10. New Delhi. 2007. Reference Source\n\nMerriam-Webster: Benefit. Merriam-Webster.com dictionary. Reference Source\n\nAbdul M, Ismail H, Mustapha M: Halal Food Certification: Case of Malaysian SME Entrepreneurs. China-USA Bus Rev. 2013; 12: 63â173.\n\nMerriam-Webster: Procedure. Merriam-Webster.com dictionary. Reference Source\n\nMerriam-Webster: Attitude. Merriam-Webster.com dictionary. Reference Source\n\nMerriam-Webster: Intention. Merriam-Webster.com Dict.\n\nAjzen I: The Theory of Planned Behavior. Organ. Behav. Hum. Decis. Process. 1991; 50: 179â211. Publisher Full Text\n\nOthman B, Shaarani S, Bahron A: The influence of knowledge, attitude and sensitivity to government policies in halal certification process on organizational performance. J. Islam. Mark. 2015; 8(3): 393â408. Publisher Full Text\n\nJaafar SN, Lalp PE, Naba MM: Consumersâ Perception, Attitudes and Purchase Intention towards Private Label Food Products in Malaysia.2.\n\nSinthamrong P, Rompho T: Factors Affecting Attitudes and Purchase Intentions Toward Branded Content on Webisodes. J. Manag. Policy Pract. 2015; 16: 64â72.\n\nYang M, Al-shaaban S, Nguyen TB: Consumer Attitude and Purchase Intention towards Organic Food A quantitative study of China. LinnÊus University; 2014.\n\nLee KH, Shin D: Consumersâ responses to CSR activities: The linkage between increased awareness and purchase intention. Public Relat. Rev. 2010; 36: 193â195. Publisher Full Text\n\nAmbali AR, Bakar AN: HalÄl food and products in Malaysia: Peopleâs awareness and policy implications. Intellect Discourse. 2013; 21: 7â32.\n\nOemar H, Prasetyaningsih E, Bakar SZA, et al.: List of questions of the questionnaire - Awareness to Register Halal Certification. figshareDataset. Epub ahead of print 2021. Publisher Full Text\n\nMenteri Hukum dan HAM (Minister of Law and Human Rights): Undang-Undang Republik Indonesia Nomor 20 Tahun 2008 (Law of Republic Indonesia Number 20/2008).2008; 1â11.\n\nHair JF Jr, Hult GTM, Ringle CM, et al.: A Primer on Partial Least Squares Structural Equation Modeling (PLS-SEM). 2nd ed. Los Angeles: SAGE; 2017.\n\nSekaran U, Bougie R: Research Method for Business. 6th ed. New York: John Wiley & Sons, Inc; 2013.\n\nBudhiasa S: Analisis Statistik Multivariate dengan Aplikasi SEM PLS SmartPLS 3.2.6. Denpasar: Udayana University Press; 2016.\n\nAjzen I: Attitudes, Personality and Behavior. New York: Open University Press; 2005.\n\nOemar H, Prasetyaningsih E, Bakar SZA, et al.: Dataset of Questionnaire Result from the respondents of Awareness to Register Halal Certification. figshareDataset. Epub ahead of print 2021. Publisher Full Text\n\nOemar H, Prasetyaningsih E, Bakar SZA, et al.: The Respondent characteristics of Awareness to Register Halal Certification. figshareDataset. Epub ahead of print 2021. Publisher Full Text"
}
|
[
{
"id": "123266",
"date": "18 Feb 2022",
"name": "Yukichika Kawata",
"expertise": [
"Reviewer Expertise applied economics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI found that the topics of this study reflect the current Indonesian status of halal certification and would attract the interest of both scholars and the general public. However, some significant issues remain unaddressed.\nMajor comments:\nM1) The procedure for constructing the sample is not appropriate. The authors state that:\nâThe questionnaires were returned by 376 people. The returned questionnaires were then sorted using predetermined criteria, yielding 137 questionnaires that met the criteria. However, due to the limitations of the software features used, this study only processed 100 questionnaires at random.â\nIt is not acceptable to select 100 out of 137 questionnaires, even if they are randomly selected. The limitations of the software may not be reasonable ground for such an exclusion because other open-source and academically-sound software such as âR\" is available, which provides packages to conduct PLS-SEM or other SEMs.\nM2) The explanations on how the final model is constructed are insufficient. For example, the authors do not report any indices of goodness-of-fit such as AIC, GFI, AGFI, and CFI.\nM3) What is the purpose of this study? The main targets of this study are micro and small-scale food enterprises, and the authors state that:\nâHence, this study aims at measuring the level of halal awareness and the intention of food MSE entrepreneurs in West Java Province, Indonesia to register halal certification.â\nI do not understand why the authors repeatedly explain the situation of exports to other countries and the status of importing countries such as the Philippines. Such descriptions seem to be redundant. For example,\nâThe globalization of the halal industry provides an opportunity for local micro and small-scale enterprises (MSEs) to sell halal products. Local MSEs, on the other hand, are hesitant to compete in the global market because they lack halal certificates, which are one of the most important requirements for entering the global market. Halal certifications are also regarded as a quality-control standard among Muslim consumers. Many non-Muslims have no qualms about eating halal food, but they may react negatively if they eat halal food accidentally and feel cheated.3 As a result, MSEs that want to enter the global market must have a halal certificate.â\nâNon-halal restaurant owners in Manila, Philippines are generally aware of the 12 halal certification standards (raw materials, tools and equipment, facilities, buildings, exterior areas, location, halal documentation, staff characteristics, staff policies, pest controls, management responsibilities, and waste management), and the majority are âWillingâ to be halal certified.â\nâHalal food certificates are required to increase self-confidence, customer trust, and customer satisfaction, although SMEs in Hat Yai, Thailand are dissatisfied with the poor dissemination of halal hub information.â\nIf the authors intend to examine the possibility of food exports by MSEs in West Java Province, the discussion section would require to be revised to meet such objectives.\nM4) The explanation of the basic information regarding food micro and small-scale enterprises (food MSEs) in West Java Province is inadequate. The authors state that:\nâAccording to our observations, the majority of micro and small-scale food entrepreneurs in West Java purchase raw materials and process them into finished products without storing and shipping.\"\nIs the authorsâ main target traditional restaurants such as âKaki lima,â âWarung,â and âRumah makanâ? Please explain the types of food MSEs in greater detail. Which of the following are included under food MSEs in this study: producers, wholesalers, retailers, distributors, or restaurants? Also, please specify or give examples of what foods (e.g., carcasses at the middle, or dressed meats at almost the final stage of the food system) MSEs mainly treat.\nMinor comments:\nS1) The authors state the following:\nâWith the issuance of âUndang-Undang Republik Indonesia No 33 Tahun 2014â (Law of Republic Indonesia No 33/2014 - English)10 regarding halal product guarantees, the halal certification has become a requirement for producers in Indonesia.â\nThey also state,\nâAlthough Islam is the majority religion in Indonesia, most micro, small, and medium enterprises (MSMEs) do not register their products for halal certification. Only 10% of MSMEs have halal certificates, according to the Association of Food and Beverage Entrepreneurs (GAPMMI) in June 2019.â\nIt appears that these two explanations are contradictory. Kindly elucidate further.\nS2) I do not agree with the following description,\nâThese indicate the food MSEs' lack of understanding and awareness of the importance of halal products.â\nIt is untrue that food MSEs lack understanding and awareness of the importance of halal products. However, they lack the necessity to obtain halal certificates. Most target local markets and may not intend to export their products to Muslims in other countries. If so, they have no incentive to acquire a halal certification for trade within Indonesia or export outside Indonesia.\nS3) Is the following extract from the paper related to âhalal awarenessâ? Since CSR is a broader concept, it might be beneficial for readers if you add an explanation of CSR in Islamic or Indonesian contexts.\nâMeanwhile, the procedure for obtaining halal certificates is relatively complex, thereby reducing the intention of SMEs to register halal certification. Meanwhile, Lee and Shin37 found that consumers' awareness of Corporate Social Responsibility (CSR) activities and purchase intentions are positively related.\"\nS4) The authors write âattitude to produce halal product (AHC)â in the body text.\nâReferring to Giyanti and Indriastiningsih,14 Waluyo,19 and Lee and Shin,37 and the Planned Behavior Theory,32 this study identifies that the variables are knowledge of halal (KH), perception of benefits (PB), perception of procedures (PP), halal awareness (HA), attitude to produce halal product (AHC) and intention to register a halal certificate.\"\nHowever, they write âAttitude to register halal productâ in Figure 1. Are they both correct?\nS5) Please add a brief explanation on \"green foods\" and possibly also on the relationship between the halal concept and green foods.\nâConsumer awareness of the green concept is a strong predictor of their intention to consume green foods,17 while customer awareness towards the halal logo fosters purchase intention.\"\nS6) Please explain the following in more detail:\nâInitially, 680 micro and small-scale food and beverage entrepreneurs in the West Java MSME community were targeted.\"\nIt is not clear if 680 is the total number of micro-and small-scale food and beverage entrepreneurs in West Java or if it is the number of entrepreneurs selected by the authors. Please consider adding the total number of micro, small, and medium food and beverage entrepreneurs in West Java in Table 1 and the number of micro and small entrepreneurs that were targets of this study.\nS7) Please specify if those involved in the pilot survey are from West Java or other provinces.\nâThe questionnaire was pretested with a small sample of food MSE entrepreneurs before being distributed to the actual respondents.\"\nS8) Please consider using \"Partial Least Squares Structural Equation Modeling (PLS-SEM)â instead of âStructural Equation Model-Partial Least Square (SEM-PLS).â\nS9) The following literature is not cited in the body text:\nâ40. Menteri Hukum dan HAM (Minister of Law and Human Rights): Undang-Undang Republik Indonesia Nomor 20 Tahun 2008 (Law of Republic Indonesia Number 20/2008).2008; 1â11.\"\nS10) Please consider quoting references for this procedure:\nâWe analyzed the chi-square of early and late respondents' responses to see if there was any potential for non-response bias (the first and last 20 percent of responses received).\"\nS11) Please also explain how many responses are considered: \"376 people\", \"137 questionnaires\", or \"100 questionnaires\"? If either 137 or 100 questionnaires are used, the results may be inaccurate if only earlier replies are considered for examination. This would hinder possible differences among respondents and may potentially cause biases.\nI also did not understand the procedure followed while gathering questionnaires. The following two descriptions appear inconsistent:\nâAs a result, the data collected reached 100 respondents in just a matter of days. Due to the limitations of the software features used, the distribution of the questionnaire was halted. As a result, all collected data was subjected to validity and reliability tests.\"\nâThe questionnaires were distributed electronically to the West Java MSME community. The questionnaires were returned by 376 people. The returned questionnaires were then sorted using predetermined criteria, yielding 137 questionnaires that met the criteria. However, due to the limitations of the software features used, this study only processed 100 questionnaires at random.\"\nAs pointed out in the major comment, I do not agree with the use of 100 samples.\nS12) Please add the corresponding population statistics to Table 7 and briefly discuss the possible biases, if any.\nâTable 7 displays the percentage of respondents for each indicator. As shown in Table 7, 98% of respondents have Islam as their religion (Muslim), 71% are female, and 88% are 26 years old or older.\"\nS13) Why do the authors not remove Christians from the selected 100 respondents? (Table 7 shows one Catholic and one Protestant.)\nS14) Please consider aligning digits in Table 8, e.g., \"3.2225\" maybe \"3.223\", etc.\nS15) Please specify the percentage selected by the authors as statistically significant (e.g., 5%) in Table 9.\nS16) Please use a period instead of a comma as a decimal point. For example, the authors used both in the following paragraph (82,6% and 82.6%, respectively).\nâBesides, the R2 value of 0.826 for HA means that 82,6% of the variance in HA is influenced by KH and PB, with values β = 0,292 and 0,683 respectively. Hence, KH and PB have a strong and significant impact on the awareness of MSE entrepreneurs about the importance of halal products. According to Hair, et al.41 R2 values of 0.75, 0.50, or 0.25 for the endogenous construct, respectively, can be described as substantial, moderate, or weak. The R2 value of an endogenous latent variable (i.e., halal awareness) described by the two predictive constructs in this study is 82.6% (see Figure 2), which is substantial.\"\nS17) The intended meaning of the following sentence is ambiguous.\nâThe finding that knowledge of halal has a significant impact on knowledge of halal is different from the finding of Giyanti and Indriastiningsih14\"\nThe phrase âknowledge of halal has a significant impact on knowledge of halalâ is not clear. Please consider rewording it.\nPlease also add a period at the end of this sentence.\nS18) I do not understand the following paragraph. Did the MSE find it beneficial or unnecessary to obtain halal certificates?\nâThe findings also show that MSEs are aware of the benefits of the halal certificate. Based on our observation, we find that they do not require a halal certificate because they have satisfied with the sales/performance that has been achieved.\"\nSimilarly, I also do not understand why the authors conclude â[h]alal certificates will therefore increase sales and revenueâ in the following context? Please consider a more logical construction of the sentence.\nâThey are unaware that halal certificates will increase consumer trust and help food businesses compete more effectively. Halal certificates will therefore increase sales and revenue.\"\nS19) Please explain the relationship between respondents of this study and âstreet vendors around Universitas Islam Bandung (Unisba)â that is mentioned in the âDiscussionâ section.\nâSocialization and training on halal awareness, halal guarantee system, and halal certification for street vendors around Universitas Islam Bandung (Unisba) which is located at Bandung, the Capital of West Java Province Indonesia have been conducted by Oemar et al.13 The trainees gain a better understanding and awareness of halal food as a result of the training. Consequently, all trainees intend to obtain a Halal Certificate.\"\nS 20) Please recheck the references carefully. For example, you can quote my work (reference 9), but the publication year, pages, etc., are incorrect.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "8690",
"date": "17 Oct 2022",
"name": "Endang Prasetyaningsih",
"role": "Author Response",
"response": "Thank you for your insightful comments. Each comment is addressed in a point-by-point format. Major comments: M1) The procedure for constructing the sample is not appropriate. The authors state that: âThe questionnaires were returned by 376 people. The returned questionnaires were then sorted using predetermined criteria, yielding 137 questionnaires that met the criteria. However, due to the limitations of the software features used, this study only processed 100 questionnaires at random.â It is not acceptable to select 100 out of 137 questionnaires, even if they are randomly selected. The limitations of the software may not be reasonable ground for such an exclusion because other open-source and academically-sound software such as âR\" is available, which provides packages to conduct PLS-SEM or other SEMs. Response: We recalculated by increasing the number of respondents from 100 to 137 in the revised manuscript. M2) The explanations on how the final model is constructed are insufficient. For example, the authors do not report any indices of goodness-of-fit such as AIC, GFI, AGFI, and CFI. Response: PLS-SEM uses SRMS, and RMStheta to test the model fit. We state that âThe PLS-SEM model fit is evaluated using standardized root mean square residual (SRMR), root mean square residual covariance (RMStheta), or the exact fit test to determine how well it predicts endogenous variables/constructs.41â We choose SRMR where the result is stated as follows âThis study uses the standardized root means square (SMSR) to assess model fit. The SMSR is 0.067, less than 0.08, indicating that the model meets the model fit criteria. Furthermore, the data can be used to estimate the model.â M3) What is the purpose of this study? The main targets of this study are micro and small-scale food enterprises, and the authors state that: âHence, this study aims at measuring the level of halal awareness and the intention of food MSE entrepreneurs in West Java Province, Indonesia to register halal certification.â I do not understand why the authors repeatedly explain the situation of exports to other countries and the status of importing countries such as the Philippines. Such descriptions seem to be redundant. For example, âThe globalization of the halal industry provides an opportunity for local micro and small-scale enterprises (MSEs) to sell halal products. Local MSEs, on the other hand, are hesitant to compete in the global market because they lack halal certificates, which are one of the most important requirements for entering the global market. Halal certifications are also regarded as a quality-control standard among Muslim consumers. Many non-Muslims have no qualms about eating halal food, but they may react negatively if they eat halal food accidentally and feel cheated.3 As a result, MSEs that want to enter the global market must have a halal certificate.â âNon-halal restaurant owners in Manila, Philippines are generally aware of the 12 halal certification standards (raw materials, tools and equipment, facilities, buildings, exterior areas, location, halal documentation, staff characteristics, staff policies, pest controls, management responsibilities, and waste management), and the majority are âWillingâ to be halal certified.â âHalal food certificates are required to increase self-confidence, customer trust, and customer satisfaction, although SMEs in Hat Yai, Thailand are dissatisfied with the poor dissemination of halal hub information.â If the authors intend to examine the possibility of food exports by MSEs in West Java Province, the discussion section would require to be revised to meet such objectives. Response: The purpose of this study is not to investigate the possibility of food exports by MSEs in West Java Province, but to assess the level of halal awareness and the intention of food producers in West Java Province, Indonesia, to register for halal certification. Therefore, we rewrite the introduction section to describe the gap between the necessity of having a halal certificate and the negative perception of halal certification among food entrepreneurs in several Indonesian cities. M4) The explanation of the basic information regarding food micro and small-scale enterprises (food MSEs) in West Java Province is inadequate. The authors state that: âAccording to our observations, the majority of micro and small-scale food entrepreneurs in West Java purchase raw materials and process them into finished products without storing and shipping.\" Is the authorsâ main target traditional restaurants such as âKaki lima,â âWarung,â and âRumah makanâ? Please explain the types of food MSEs in greater detail. Which of the following are included under food MSEs in this study: producers, wholesalers, retailers, distributors, or restaurants? Also, please specify or give examples of what foods (e.g., carcasses at the middle, or dressed meats at almost the final stage of the food system) MSEs mainly treat. Response: We revised the statement to be: âThe study was carried out in West Java Province, Indonesia. The population is the MSE food producers listed in the Central Bureau of Statistics of West Java, such as producers of cassava chips, shredded catfish, âbagelenâ cakes, chocolate ârangginangâ, candied vegetables, market snacks, and so on.â Minor comments: S1) The authors state the following: âWith the issuance of âUndang-Undang Republik Indonesia No 33 Tahun 2014â (Law of Republic Indonesia No 33/2014 - English)10 regarding halal product guarantees, the halal certification has become a requirement for producers in Indonesia.â They also state, âAlthough Islam is the majority religion in Indonesia, most micro, small, and medium enterprises (MSMEs) do not register their products for halal certification. Only 10% of MSMEs have halal certificates, according to the Association of Food and Beverage Entrepreneurs (GAPMMI) in June 2019.â It appears that these two explanations are contradictory. Kindly elucidate further. Response: We rewrite the introduction section and describe the contradiction. âIndonesia is a country where Islam is the majority religion. The Indonesian government has mandated that food producers have halal certificates to protect consumers, particularly Muslim consumers when purchasing food, by issuing Undang-Undang Republik Indonesia No 33 Tahun 2014\" (Law of Republic Indonesia No 33/2014 - English)10 regarding halal product guarantees. However, statistics show that the majority of food micro, small, and medium-scale enterprises (food MSMEs) do not register their products for halal certification. According to the Association of Food and Beverage Entrepreneurs (GAPMMI) in June 2019, only 10% of MSMEs have halal certificates.10 This contradiction indicates that the intention of SMEs to sell halal-certified products remains low. They may be unaware of the benefits of halal certification or have a negative perception of halal certificates. Several studies on the perception of halal certificates have been conducted in various cities in Indonesia.â S2) I do not agree with the following description,âThese indicate the food MSEs' lack of understanding and awareness of the importance of halal products.â It is untrue that food MSEs lack understanding and awareness of the importance of halal products. However, they lack the necessity to obtain halal certificates. Most target local markets and may not intend to export their products to Muslims in other countries. If so, they have no incentive to acquire a halal certification for trade within Indonesia or export outside Indonesia. Response: Focus on the purpose of our research; we do not discuss product export; rather, we measure awareness and intention to register for a halal certificate. Several studies on food entrepreneurs' perceptions of halal certification in several Indonesian cities are as follows. âThe majority of MSME entrepreneurs in some cities of East Java Province, Indonesia, such as Bangkalan, Pamekasan, and Pasuruan, understand the significance of halal certificates. However, they consider halal certification to be unimportant.12 In East Kalimantan Province, Indonesia the MSME entrepreneurs even underestimate the halal certificate due to believing that their business is running smoothly despite the lack of a halal certificate.13 Meanwhile, entrepreneurs in other Indonesian cities such as the Greater Jakarta Area,14 Malang City East Java Province,15 and Surakarta City Central Java Province16 did not register halal certification because they perceive the process as difficult and costly. These cases indicate MSME entrepreneursâ lack of necessity for obtaining halal certificates. â S3) Is the following extract from the paper related to âhalal awarenessâ? Since CSR is a broader concept, it might be beneficial for readers if you add an explanation of CSR in Islamic or Indonesian contexts. âMeanwhile, the procedure for obtaining halal certificates is relatively complex, thereby reducing the intention of SMEs to register halal certification. Meanwhile, Lee and Shin37 found that consumers' awareness of Corporate Social Responsibility (CSR) activities and purchase intentions are positively related.\" Response: We concentrate on the findings of the Lee and Shin24 study, specifically the relationship between awareness and intention, rather than the case study. Hence, we updated the explanation as follows: âAccording to Liba et al.,9 Elias et al.,20 and Masithoh et al.,21 awareness of halal is positively correlated with the intention to obtain halal certification. Meanwhile, Dinev and Hu,22â25 Bachok et al.,23 Lee and Shin,24 Rezai et al.,25 state that customer awareness is a strong predictor of a customer's intent to buy or select a product. These indicate that awareness influences intention.â S4) The authors write âattitude to produce halal product (AHC)â in the body text. âReferring to Giyanti and Indriastiningsih,14 Waluyo,19 and Lee and Shin,37 and the Planned Behavior Theory,32 this study identifies that the variables are knowledge of halal (KH), perception of benefits (PB), perception of procedures (PP), halal awareness (HA), attitude to produce halal product (AHC) and intention to register a halal certificate.\" However, they write âAttitude to register halal productâ in Figure 1. Are they both correct? Response: We revised the explanation and Figure 1. S5) Please add a brief explanation on \"green foods\" and possibly also on the relationship between the halal concept and green foods. âConsumer awareness of the green concept is a strong predictor of their intention to consume green foods,17 while customer awareness towards the halal logo fosters purchase intention.\" Response: We are interested in the study's findings, specifically the relationship between awareness and intention, rather than the relationship between the halal concept and green foods. As a result, we revised the explanation as follows: âConsumer awareness is a strong predictor of their intention to consume/purchase foods.23,25â S6) Please explain the following in more detail: âInitially, 680 micro and small-scale food and beverage entrepreneurs in the West Java MSME community were targeted.\" It is not clear if 680 is the total number of micro-and small-scale food and beverage entrepreneurs in West Java or if it is the number of entrepreneurs selected by the authors. Please consider adding the total number of micro, small, and medium food and beverage entrepreneurs in West Java in Table 1 and the number of micro and small entrepreneurs that were targets of this study. Response: We revise the explanation as follows: âThe convenience sampling technique was used in this study. The total food producer of MSE listed in the Central Bureau of Statistics of West Java was estimated to be around 2300 people. We worked with \"Sahabat UMKM Jawa Barat\" (West Java MSME association-English), a local association of micro, small, and medium-scale entrepreneurs who are engaged in a variety of fields such as culinary, fashion, crafts, and other businesses or industries. The local association has over 1000 members, with 68 percent of them being food and beverage entrepreneurs, or approximately 680 entrepreneurs. This study's sample consists of food producers who meet the predetermined criteria among the 680 entrepreneurs.â S7) Please specify if those involved in the pilot survey are from West Java or other provinces. âThe questionnaire was pretested with a small sample of food MSE entrepreneurs before being distributed to the actual respondents.\" Response: We included the pilot survey participant in the following statement: âThe questionnaire was pretested with a small sample of members of the West Java MSME association before being distributed to the actual respondents.â S8) Please consider using \"Partial Least Squares Structural Equation Modeling (PLS-SEM)â instead of âStructural Equation Model-Partial Least Square (SEM-PLS).â Response: As suggested, we have revised SEM-PLS to PLS-SEM. S9) The following literature is not cited in the body text: â40. Menteri Hukum dan HAM (Minister of Law and Human Rights): Undang-Undang Republik Indonesia Nomor 20 Tahun 2008 (Law of Republic Indonesia Number 20/2008).2008; 1â11.\" Response: We have quoted \"Undang-Undang Republik Indonesia Nomor 20/2008â (Law of the Republic of Indonesia Number 20/2008).39 in body text and Table 2 as follows: âCategorization of business scale refers to the \"Undang-Undang Republik Indonesia Nomor 20/2008â (Law of the Republic of Indonesia Number 20/2008).39â âTable 2. Category of micro, small and medium business in Indonesia according to Law of the Republic of Indonesia No. 20/2008.39â S10) Please consider quoting references for this procedure: âWe analyzed the chi-square of early and late respondents' responses to see if there was any potential for non-response bias (the first and last 20 percent of responses received).\" Response: We discovered an appropriate reference in our research and changed the statement to: âWe examined the responses of early and late respondents to see if there was any possibility of non-response bias. According to Lindner et al.,44 respondents were classified into two groups: early and late respondents. Late respondents were operationally and arbitrarily defined as the last half of respondents. Because there were 137 respondents in this study, 69 were identified as early respondents and 68 as late respondents. Hence, the independent samples t-test was then used to compare the two groups' responses to Likert scale questions. The findings show that there is no significant difference in key metrics responses between early and late respondents.â S11) Please also explain how many responses are considered: \"376 people\", \"137 questionnaires\", or \"100 questionnaires\"? If either 137 or 100 questionnaires are used, the results may be inaccurate if only earlier replies are considered for examination. This would hinder possible differences among respondents and may potentially cause biases. I also did not understand the procedure followed while gathering questionnaires. The following two descriptions appear inconsistent: âAs a result, the data collected reached 100 respondents in just a matter of days. Due to the limitations of the software features used, the distribution of the questionnaire was halted. As a result, all collected data were subjected to validity and reliability tests.\" âThe questionnaires were distributed electronically to the West Java MSME community. The questionnaires were returned by 376 people. The returned questionnaires were then sorted using predetermined criteria, yielding 137 questionnaires that met the criteria. However, due to the limitations of the software features used, this study only processed 100 questionnaires at random.\" Response: We revised the explanation as follows: âThe questionnaires were distributed to the members of the West Java MSME association's WhatsApp groups. The questionnaires were returned by 376 respondents. The returned questionnaires were then sorted using predetermined criteria, yielding 137 respondents that met the criteria.â âFinally, we decided to distribute the electronic questionnaire Google Forms in May 2020 to the West Java MSME association's WhatsApp groups. In just a few days, the data collected reached 137 respondents who met the predetermined criteria, so all collected data were subjected to validity and reliability tests.â As pointed out in the major comment, I do not agree with the use of 100 samples. S12) Please add the corresponding population statistics to Table 7 and briefly discuss the possible biases, if any. âTable 7 displays the percentage of respondents for each indicator. As shown in Table 7, 98% of respondents have Islam as their religion (Muslim), 71% are female, and 88% are 26 years old or older.\" Response: We tested the non-biased response, and the results are as follows: âWe examined the responses of early and late respondents to see if there was any possibility of non-response bias. According to Lindner et al.,44 respondents were classified into two groups: early and late respondents. Late respondents were operationally and arbitrarily defined as the last half of respondents. Because there were 137 respondents in this study, 69 were identified as early respondents and 68 as late respondents. Hence, the independent samples t-test was then used to compare the two groups' responses to Likert scale questions. The findings show that there is no significant difference in key metrics responses between early and late respondents.â S13) Why do the authors not remove Christians from the selected 100 respondents? (Table 7 shows one Catholic and one Protestant.) Response: We state that âNon-Muslim respondents are involved because producing halal food regardless of the religion of the food producers.â S14) Please consider aligning digits in Table 8, e.g., \"3.2225\" maybe \"3.223\", etc. Response: We have revised the numbers S15) Please specify the percentage selected by the authors as statistically significant (e.g., 5%) in Table 9. Response: We add the significance level of 5% in the body text and Table 12. âBy using a significance level of 5%, the path coefficient will be significant if the t-value is larger than 1.65.48â S16) Please use a period instead of a comma as a decimal point. For example, the authors used both in the following paragraph (82,6% and 82.6%, respectively). âBesides, the R2 value of 0.826 for HA means that 82,6% of the variance in HA is influenced by KH and PB, with values β = 0,292 and 0,683 respectively. Hence, KH and PB have a strong and significant impact on the awareness of MSE entrepreneurs about the importance of halal products. According to Hair, et al.41 R2 values of 0.75, 0.50, or 0.25 for the endogenous construct, respectively, can be described as substantial, moderate, or weak. The R2 value of an endogenous latent variable (i.e., halal awareness) described by the two predictive constructs in this study is 82.6% (see Figure 2), which is substantial.\" Response: We use a period as a decimal point in the following paragraph âBesides, the R2 value of 0.841 for HA means that 84.1% of the variance in HA is influenced by KH and PB, with values β = 0.169 and 0.791 respectively. Hence, KH and PB have a strong and significant impact on the halal awareness of MSE food producers.â âAccording to Hair, et al.,41  R2 values of 0.75, 0.50, or 0.25 for the endogenous construct, respectively, can be described as substantial, moderate, or weak. The R2 value of an endogenous latent variable (i.e., halal awareness) described by the two predictive constructs in this study is 84.1% (see Figure 3), which is substantial.â S17) The intended meaning of the following sentence is ambiguous. âThe finding that knowledge of halal has a significant impact on knowledge of halal is different from the finding of Giyanti and Indriastiningsih14\" The phrase âknowledge of halal has a significant impact on knowledge of halalâ is not clear. Please consider rewording it. Please also add a period at the end of this sentence. Response: We revised the explanation as follows: âThe finding that knowledge of halal has a significant impact on halal awareness is different from the finding of Giyanti and Indriastiningsih.16â S18) I do not understand the following paragraph. Did the MSE find it beneficial or unnecessary to obtain halal certificates? âThe findings also show that MSEs are aware of the benefits of the halal certificate. Based on our observation, we find that they do not require a halal certificate because they have satisfied with the sales/performance that has been achieved.\" Response: The explanation has been revised as follows: âThe findings also indicate that MSE food producers are aware of the benefits of the halal certificate. However, our observation revealed that they perceive obtaining a halal certificate as prohibitively expensive. It will cause the halal-certified product's selling price to rise. They are concerned that increasing selling prices will reduce sales. As a result, they perceive that obtaining a halal certificate is unnecessary. In any case, their business is running smoothly without the halal certificate. It aligns with the findings of Prabowo et al.13â Similarly, I also do not understand why the authors conclude â[h]alal certificates will therefore increase sales and revenueâ in the following context? Please consider a more logical construction of the sentence. âThey are unaware that halal certificates will increase consumer trust and help food businesses compete more effectively. Halal certificates will therefore increase sales and revenue.\" Response: We delete this statement due to the contradiction with the previous statement. S19) Please explain the relationship between respondents of this study and âstreet vendors around Universitas Islam Bandung (Unisba)â that is mentioned in the âDiscussionâ section. âSocialization and training on halal awareness, halal guarantee system, and halal certification for street vendors around Universitas Islam Bandung (Unisba) which is located at Bandung, the Capital of West Java Province Indonesia have been conducted by Oemar et al.13 The trainees gain a better understanding and awareness of halal food as a result of the training. Consequently, all trainees intend to obtain a Halal Certificate.\" Response: The explanation has been revised as follows. âOemar et al.17 proved that socialization and training on halal awareness, halal assurance systems, and halal certification increase the understanding and awareness about halal-certified food so that all participants intend to obtain a Halal Certificate after completing the training.â S 20) Please recheck the references carefully. For example, you can quote my work (reference 9), but the publication year, pages, etc., are incorrect. Response: The reference writing has been corrected."
}
]
},
{
"id": "127721",
"date": "31 Mar 2022",
"name": "Ai Chin Thoo",
"expertise": [
"Reviewer Expertise marketing and supply chain management"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe practical gaps are not aligned with the research objectives. What is the relationship between the global market and the intention of local MSEs for the halal certificates? Need to include practical and theoretical gaps in the Introduction part.\nThe literature review is not coherently discussed. Lack of justifications for the hypotheses development.\n\nThe population, sample, sample size, sampling technique, and data collection procedure need to be highlighted and discussed in detail.\n\nPlease justify why 100 samples were used for data analysis. 'The questionnaires were distributed electronically to the West Java MSME community. The questionnaires were returned by 376 people. The returned questionnaires were then sorted using predetermined criteria, yielding 137 questionnaires that met the criteria. However, due to the limitations of the software features used, this study only processed 100 questionnaires at random.'\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "8691",
"date": "17 Oct 2022",
"name": "Endang Prasetyaningsih",
"role": "Author Response",
"response": "Thank you for your insightful comments. Each comment is addressed in a point-by-point format. 1. The practical gaps are not aligned with the research objectives. What is the relationship between the global market and the intention of local MSEs for the halal certificates? Need to include practical and theoretical gaps in the Introduction part. Response: We have rewritten the introduction section. The purpose of this study is to assess the level of halal awareness and the intention of food producers in West Java Province Indonesia to register for halal certification. Hence, we rewrite the introduction section to describe the gap between the necessity of having a halal certificate and the negative perception of halal certification among food entrepreneurs in several Indonesian cities. 2. The literature review is not coherently discussed. Lack of justifications for the hypotheses development. Response: We have rewritten the literature review section 3. The population, sample, sample size, sampling technique, and data collection procedure need to be highlighted and discussed in detail. Response: We have added a new sub-section called \"Population and Sample.\" and the data collectionâs description.  âPopulation and sample The study was carried out in West Java Province, Indonesia. The population is the MSE food producers listed in the Central Bureau of Statistics of West Java, such as producers of cassava chips, shredded catfish, âbagelenâ cakes, chocolate ârangginangâ, candied vegetables, market snacks, and so on. Categorization of business scale refers to the \"Undang-Undang Republik Indonesia Nomor 20/2008â (Law of the Republic of Indonesia Number 20/2008).39 Table 2 shows the categorization of the business scale. The population chosen consisted of those who met the criteria 1) a food producer of micro and small-scale with annual sales turnover of fewer than 2,500 Rupiahs and net assets of fewer than 500 million Rupiahs; 2) have an ongoing business, and 3) no halal certificate.â The convenience sampling technique was used in this study. The total food producer of MSE listed in the Central Bureau of Statistics of West Java was estimated to be around 2300 people. We worked with \"Sahabat UMKM Jawa Barat\" (West Java MSME association-English), a local association of micro, small, and medium-scale entrepreneurs who are engaged in a variety of fields such as culinary, fashion, crafts, and other businesses or industries. The local association has over 1000 members, with 68 percent of them being food and beverage entrepreneurs, or approximately 680 entrepreneurs. This study's sample consists of food producers who meet the predetermined criteria among the 680 entrepreneurs.â âData collection A questionnaire is chosen as the research instrument. The questionnaires were re-translated from English to Indonesian, except for those references that were already in Indonesian. Each variable is made up of measurement items which are scored on a Likert scale of 1 (strongly disagree) to 5 (strongly agree). A Google Form with a cover letter and a set of questionnaires were sent out to the potential respondents who are members of the West Java MSME association WhatsApp groups. This method was chosen because of the COVID-19 pandemic outbreak. In addition, the designed questionnaires could be collected without conducting direct visits to the respondents. The respondents could not participate in the survey unless they gave their written consent. Data were collected from March to May 2020. A copy of the distributed questionnaires can be found in Extended Data.40 The questionnaire was pretested with a small sample of members of the West Java MSME association before being distributed to the actual respondents. Based on pretest feedback, the wording of some items was refined and modified to guarantee that the validity and reliability of each variable meet the required standard. The question items were scored on a Likert scale of 1 (strongly disagree) to 5 (strongly agree). The follow-up of this plan is described later in the data preparation section. The convenience sampling method is used in the following manner. Distribute questionnaires, Wait for responses to the distributed questionnaire, Collect data until the sample count is adequate. Screen participants based on the criteria specified.â 4. Please justify why 100 samples were used for data analysis. 'The questionnaires were distributed electronically to the West Java MSME community. The questionnaires were returned by 376 people. The returned questionnaires were then sorted using predetermined criteria, yielding 137 questionnaires that met the criteria. However, due to the limitations of the software features used, this study only processed 100 questionnaires at random.' Response: In the revised manuscript, we recalculated by increasing the number of respondents from 100 to 137 based on the reviewers' recommendations."
}
]
}
] | 1
|
https://f1000research.com/articles/11-170
|
https://f1000research.com/articles/12-348/v1
|
29 Mar 23
|
{
"type": "Data Note",
"title": "The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence",
"authors": [
"Riham Ayoubi",
"Ian McDowell",
"Maryam Fotouhi",
"Kathleen Southern",
"Peter S. McPherson",
"Carl Laflamme",
"NeuroSGC/YCharOS/EDDU collaborative group",
"ABIF Consortium",
"Riham Ayoubi",
"Ian McDowell",
"Maryam Fotouhi",
"Kathleen Southern",
"Peter S. McPherson"
],
"abstract": "Profilin-1, a member of the Profilin family, is a ubiquitously expressed protein that controls actin polymerization in a concentration-dependent manner. As mutations in the Profilin-1 gene have potential implications in neurodegenerative disease progression, well-characterized anti-Profilin-1 antibodies would be beneficial to the scientific community. In this study, we characterized sixteen Profilin-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence applications, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.",
"keywords": [
"Uniprot ID P07737",
"PFN1",
"Profilin-1",
"antibody characterization",
"antibody validation",
"Western blot",
"immunoprecipitation",
"immunofluorescence"
],
"content": "Introduction\n\nProfilin-1, encoded by the PFN1 gene, is ubiquitously expressed and the most abundant of the Profilin genes.1 It plays an essential role in the polymerization of actin by binding and sequestering actin monomers, indicating its cytoskeletal function.2,3 At high concentrations, Profilin-1 act as an inhibitor of actin polymerization whereas at low concentrations, it acts as a catalyst.3,4\n\nProfilin-1 is of recent interest thanks to the identification of PFN1 mutations in 25 human familial amyotrophic lateral sclerosis (fALS) patients.5â8 PFN1 is among a group of ALS-related genes that directly affect cytoskeletal dynamics, namely TUBA4, ALS2, KIF5A and SPAST, hypothesizing that cytoskeletal dysfunction contributes to motor neuron degeneration.9 PFN1 mutant mice that carry the G118V mutation have motor defects consistent with ALS pathology, suggesting that Profilin-1 molecular studies would provide insight into pathogenic mechanisms of motor neuron disease.10 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.\n\nHere, we compared the performance of a range of high-quality commercially available antibodies for Profilin-1 and characterized several antibodies for Western blot, immunoprecipitation and immunofluorescence experiments, enabling biochemical and cellular assessment of Profilin-1 properties and function.\n\n\nResults and discussion\n\nOur standard protocol involved comparing readouts from wild-type (WT) and knockout (KO) cells.11â13 The first step was to identify a cell line(s) that expressed sufficient levels of Profilin-1 to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million âTPMâ + 1), which we found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Of all cell lines analyzed, commercially available HAP1 cells expressed the PFN1 RNA transcript above the adequate cut-off level. Parental and PFN1 KO HAP1 cells were obtained from Horizon Discovery (Table 1).\n\nFor Western blot experiments, we resolved proteins from WT and PFN1 KO cell extracts and probed them side-by-side with all antibodies in parallel12,13 (Figure 1).\n\nLysates of HAP1 (WT and PFN1 KO) were prepared and 50 ÎŒg of protein were processed for Western blot with the indicated Profilin-1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the polyacrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exception was given for antibody A9188** which was titrated to 1/30000, as the signal was too weak when following the supplierâs recommendation. When the concentration was not indicated by the supplier, which was the case for AC-PFN1-4**, we tested the antibody at 1/1000. Antibody dilution used: 11680-1-AP at 1/6000, 67390-1-lg* at 1/30000, MAB7779* at 1/5000, NBP2-59778* at 1/1000, NBP2-67078* at 1/2000, GTX102072 at 1/3000, 3246** at 1/1000, A9188 at 1/30000, AC-PFN1-4** at 1/1000, ARP48269 at 1/2000, MA5-25113* at 1/2000, MA5-25120* at 1/2000, MA5-32683** at 1/2000, ab242369* at 1/2000, ab133529* at 1/10000, ab124904** at 1/30000. Predicted band size: 15 kDa. *Monoclonal antibody, **Recombinant antibody.\n\nFor immunoprecipitation experiments, we used the antibodies to immunopurify Profilin-1 from HAP1 WT cell extracts. The performance of each antibody was evaluated by detecting the Profilin-1 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates12,13 (Figure 2).\n\nHAP1 lysates were prepared, and IP was performed using 2.0 ÎŒg of the indicated Profilin-1 antibodies pre-coupled to Dynabeads protein G or protein A or Flag-M2 magnetic beads. Samples were washed and processed for Western blot with the indicated Profilin-1 antibody. For Western blot, MA5-25120* was used at 1/5000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=2% starting material; UB=2% unbound fraction; IP=Immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.\n\nFor immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.12â14 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).\n\nHAP1 WT and PFN1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well glass plate. Cells were stained with the indicated Profilin-1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. Primary antibodies were tested at 1.0 ÎŒg/mL. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 67390-1-Ig*, NBP2-67078*, MA5-25113*, MA5-25120* and MA5-32683** which were titrated as the signals were too weak when following the supplierâs recommendations. When the concentrations were not indicated by the supplier, which was the case for antibodies 11680-1-AP, MAB7779*, NBP2-59778*, 3246**, A9188**, ARP48269, ab242369* and ab133529*, we tested antibodies at 1/400, 1/1000, 1/500, 1/100, 1/3000,1/500, 1/500 and 1/60, respectively. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: 11680-1-AP at 1/400, 67390-1-lg* at 1/2000, MAB7779* at 1/1000, NBP2-59778* at 1/500, NBP2-67078* at 1/1000, GTX102072 at 1/500, 3246** at 1/100, A9188** at 1/3000, ARP48269 at 1/500, MA5-25113* at 1/1000, MA5-25120* at 1/600, MA5-32683** at 1/1000, ab242369* at 1/500, ab133529* at 1/60, ab124904** at 1/150. Bars=10 ÎŒm. *Monoclonal antibody, **Recombinant antibody.\n\nIn conclusion, we have screened Profilin-1 commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies using our standardized experimental conditions. The underlying data can be found on Zenodo.15,16\n\n\nMethods\n\nAll Profilin-1 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure the antibodies are cited properly.17 Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Peroxidase-conjugated monoclonal anti-Flag M2 is from MilliporeSigma (cat. number A8592). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429). The anti-FLAG (M2 clone) conjugated with Cy3 is from MilliporeSigma (cat. number A9594).\n\n* Monoclonal antibody.\n\n** Recombinant antibody.\n\n1 Refer to RRID recently added to the Antibody Registry (on January 2023), they will be available on the Registry website in coming weeks.\n\na AC-PFN1-4** sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFYSSYMYWVRQAPGKGLEWVSSISGYGGYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWDGYMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWDYGLPTFGQGTKLEIK\n\nhttps://www.eubopen.org/antibodies.\n\nBoth HAP1 WT and PFN1 KO cell lines used are listed in Table 1, together with their corresponding Research Resource Identifiers (RRID), to ensure the cell lines are cited properly.18 Cells were cultured in DMEM high glucose (GE Healthcare, cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent, cat. number 609065), 100 IU penicillin and 100 ÎŒg/mL streptomycin (Wisent, cat. number 450201).\n\nWestern blots were performed as described in our standard operating procedure.19 HAP1 WT and PFN1 KO were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat number 0089901) supplemented with 1à protease inhibitor cocktail mix (MilliporeSigma, cat. number 78429). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000à g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western blot. BLUelf prestained protein ladder (GeneDireX, cat. number PM008-0500) was used.\n\nWestern blots were performed with precast midi 10% Bis-Tris polyacrylamide gels (Thermo Fisher Scientific, cat. number WG1201BOX) ran with MES SDS buffer (Thermo Fisher Scientific, cat. number NP000202), loaded in LDS sample buffer (Thermo Fisher Scientific, cat. number NP0008) with 1à sample reducing agent (Thermo Fisher Scientific, cat. number NP0009) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show them together with individual Western blots. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095) in TBS with 0.1% Tween 20 (TBST) (Cell Signalling, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 ÎŒg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) prior to detection with the iBright⢠CL1500 Imaging System from Thermo Fisher Scientific (cat. number A44240).\n\nImmunoprecipitation was performed as described in our standard operating procedure.20 Antibody-bead conjugates were prepared by adding 2 ÎŒg or 20 ÎŒL of antibody at an unknown concentration to 500 ÎŒL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a 1.5 mL microcentrifuge tube, together with 30 ÎŒL of Dynabeads protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively) or anti-Flag M2 magnetic beads from MilliporeSigma (cat. number M8823). Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.\n\nHAP1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) from Thermo Fisher Scientific (cat. number 87788), supplemented with protease inhibitor from MilliporeSigma (cat. number P8340). Lysates were rocked 30 min at 4°C and spun at 110,000à g for 15 min at 4°C. 0.5 mL aliquots at 2.0 mg/mL of lysate were incubated with an antibody-bead conjugate for ~1 hr at 4°C. Following centrifugation, the unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP lysis buffer and processed for SDS-PAGE and Western blot on a precast midi 10% Bis-Tris polyacrylamide gels as described above.\n\nImmunofluorescence was performed as described in our standard operating procedure.12â14 HAP1 WT and TMEM106B KO were labelled with CellTrackerTM green (Thermo Fisher Scientific, cat. number C2925) or CellTrackerTM deep red (Thermo Fisher Scientific, cat. number C34565) fluorescence dye, respectively. The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. number D3571) fluorescent stain. WT and KO cells were plated in 96 well glass plates (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37oC, 5% CO2. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed three times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary Profilin-1 antibodies overnight at 4°C. Cells were then washed three times for 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 ÎŒg/mL for 1 hr at room temperature with DAPI. Cells were washed three times for 10 min with IF buffer and once with PBS.\n\nImages were acquired on an ImageXpress micro confocal high-content microscopy system (Molecular Devices), using a 20à NA 0.95 water immersion objective and scientific CMOS cameras (16-bit, 1.97mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm, 692/40 nm) to excite DAPI, CellTrackerTM Green, Alexa fluor 555 and CellTrackerTM Red, respectively. Images had pixel sizes of 0.68 à 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity, ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaXpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTrackerTM channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the âcytoâ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns.21 Masks were used to generate cell outlines for intensity quantification. Background was subtracted using a minimum projection of all images collected in the Alexa fluor 555 channel. Images shown for the Alexa fluor 555 channel were modified by applying a Gaussian blur filter with a radius value of one pixel. Figures were first assembled with Adobe Photoshop (version 24.2.1) to adjust contrast then finally assembled with Adobe Illustrator (version 27.3.1).",
"appendix": "Data availability\n\nZenodo: Antibody Characterization Report for Profilin-1, https://doi.org/10.5281/zenodo.7249258. 15\n\nZenodo: Dataset for the Profilin-1 antibody screening study, https://doi.org/10.5281/zenodo.7671118. 16\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nAcknowledgment\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Ian McDowell, Peter S. McPherson, Chetan Raina, Wolfgang Reintsch and Kathleen Southern\n\nABIF consortium: Claire M. Brown and Joel Ryan\n\n\nReferences\n\nDing Z, Bae YH, Roy P: Molecular insights on context-specific role of profilin-1 in cell migration. Cell Adhes. Migr. 2012; 6(5): 442â534. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarlsson L, Nyström LE, Sundkvist I, et al.: Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells. J. Mol. Biol. 1977; 115(3): 465â483. PubMed Abstract | Publisher Full Text\n\nAlkam D, Feldman EZ, Singh A, et al.: Profilin1 biology and its mutation, actin(g) in disease. Cell. Mol. Life Sci. 2017; 74(6): 967â981. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYarmola EG, Bubb MR: How depolymerization can promote polymerization: the case of actin and profilin. Bioessays. 2009; 31(11): 1150â1160. PubMed Abstract | Publisher Full Text\n\nIngre C, Landers JE, Rizik N, et al.: A novel phosphorylation site mutation in profilin 1 revealed in a large screen of US, Nordic, and German amyotrophic lateral sclerosis/frontotemporal dementia cohorts. Neurobiol. Aging. 2013; 34(6): 1708.e1â1708.e6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith BN, Vance C, Scotter EL, et al.: Novel mutations support a role for Profilin 1 in the pathogenesis of ALS. Neurobiol. Aging. 2015; 36(3): 1602.e17â1602.e27. Publisher Full Text\n\nWu CH, Fallini C, Ticozzi N, et al.: Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature. 2012; 488(7412): 499â503. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen Y, Zheng ZZ, Huang R, et al.: PFN1 mutations are rare in Han Chinese populations with amyotrophic lateral sclerosis. Neurobiol. Aging. 2013; 34(7): 1922.e1â1922.e5. Publisher Full Text\n\nCastellanos-Montiel MJ, Chaineau M, Durcan TM: The Neglected Genes of ALS: Cytoskeletal Dynamics Impact Synaptic Degeneration in ALS. Front. Cell. Neurosci. 2020; 14: 594975. Publisher Full Text\n\nFil D, DeLoach A, Yadav S, et al.: Mutant Profilin1 transgenic mice recapitulate cardinal features of motor neuron disease. Hum. Mol. Genet. 2017; 26(4): 686â701. PubMed Abstract | Publisher Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nAlshafie W, Ayoubi R, Fotouhi M, et al.: The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence [version 1; peer review: awaiting peer review]. F1000Res. 2023; 2023(12): 172.\n\nAlshafie W, McPherson P, Laflamme C: Antibody screening by Immunofluorescence.2021.\n\nMcDowell I, Ayoubi R, Ryan J, et al.: Antibody Characterization Report for Profilin-1.2022. Publisher Full Text\n\nLaflamme C: Dataset for the Profilin-1 antibody screening study. [Data set]. Zenodo. 2023. Publisher Full Text\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358âD367. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25â38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAyoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021.\n\nAyoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021.\n\nStringer C, Wang T, Michaelos M, et al.: Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021; 18(1): 100â106. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "168349",
"date": "14 Apr 2023",
"name": "Partha Roy",
"expertise": [
"Reviewer Expertise cell biology",
"cancer biology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a well-executed study, and a good resource for researchers working on Pfn1.\n\nI would have liked to see some conclusions with regard to the recommendation of antibodies for specific purposes based on the findings of the study.\n\nTable 2: concentration is listed as microgram/microliter - should it not be microgram/ml?\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "9571",
"date": "21 Apr 2023",
"name": "Kathleen Southern",
"role": "Author Response",
"response": "Thank you to Partha Roy (reviewer #1) for your review and further contribution to this study for Profilin-1. Dr. Roy believes our study would be improved if we drew conclusions from the data and provided antibody recommendations for the different applications. After presenting the YCharOS initiative, which aims to characterize antibodies for human proteins to dissolve the reproducibility crisis, on several occasions, weâve found that for the most part, scientists interested in our reports have the expertise to interpret the antibody characterization data. Moreover, we do not score/recommend antibodies because we tested the antibodies under one set of conditions, and the scoring/recommendation is only valid under this precise experimental setup and in the cell line used. That being said, YCharOS reports serve as an invaluable guide pointing scientists to appropriate antibodies for their experimental needs. Dr. Roy also asks whether the concentration units listed in Table 2 should be in mg/mL rather than ÎŒg/ÎŒl. To that we would like to elucidate the fact that both units of concentration are equivalent. Using mg/mL is equivalent to using ÎŒg/ÎŒl. Thank you again to Partha Roy for your suggestions!"
},
{
"c_id": "9960",
"date": "29 Aug 2023",
"name": "Kathleen Southern",
"role": "Author Response",
"response": "As a follow-up from our previous response, we would like to clarify that the antibody concentrations displayed in Table 2 are converted to the same unit of concentration, which we chose to be microgram/microliter. In some cases, yes the concentrations provided by the supplier are in microgram/milileter but we convert the concentrations to microgram/microliter to provide consistency with our units across all the tested antibodies. Along with our response regarding your suggestion to provide conclusions regarding antibody performance, we hope we clarified your reservations."
}
]
},
{
"id": "183160",
"date": "18 Jul 2023",
"name": "Jieya Shao",
"expertise": [
"Reviewer Expertise cell biology",
"cancer biology",
"genome integrity maintenance"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an excellent study containing high-quality data and detailed experimental procedures. The paper is clearly written and figures are nicely presented with high levels of technical detail allowing replication by others. The findings are highly informative for investigators interested in profilin-1, particularly with regard to its antibodies for immunofluorescence staining and immunoprecipitation purposes.\nThe only suggestion would be to clarify the purpose of using the anti-FLAG M2 magnetic beads for immunoprecipitation experiments. Which antibodies were characterized using these beads and why?\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "9961",
"date": "29 Aug 2023",
"name": "Kathleen Southern",
"role": "Author Response",
"response": "Thank you Jieya Shao for your response to this report. Antibody AC-PFN1-4, donated by the Structural Genomics Consortium (SGC), was characterized in the Immunoprecipitation experiment using the anti-FLAG M2 magnetic beads. The reason being that the antibodies produced at the SGC are recombinant renewable antibodies (Fabs, IgGs or scFv-Fv fusions) that contain the FLAG fusion peptide sequence. That being said, anti-FLAG M2 magnetic beads are required to detect the FLAG tag on the antibody and immunoprecipitate the target antigen of interest. For more information, visit the SGC website and Millipore Sigma product sheet."
}
]
},
{
"id": "178173",
"date": "10 Aug 2023",
"name": "Xiaoyan Du",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article described a work to identify high-performing antibodies for Profilin-1 using 16 Profilin-1 commercial antibodies by Western blot, immunoprecipitation and immunofluorescence analysis. It is a very significant job for those who investigate PFN1 function in medical and biological field. I concerned following issues which need explanation by authors:\nAll WB experiments are lack of reference such as GAPDH etc. when tested PFN1. Therefore, the readers could not know if the difference among the bands detected by each kind of antibodies caused by various commercial antibodies or the technique.\n\nAs to test antibodies, at least two kind of cell lines are needed. The introduction should give readers more information about PFN1 function in more diseases, not just focus on neurodegenerative disease.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-348
|
https://f1000research.com/articles/11-593/v1
|
01 Jun 22
|
{
"type": "Genome Note",
"title": "Whole genome sequencing data of Candida krusei, pathogen causing Candidaemia, from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia",
"authors": [
"Fadilah Fadilah",
"Robiatul Adawiyah",
"Retno Wahyuningsih",
"Anna Rozaliyani",
"Ayu Eka Fatril",
"Rafika Indah Paramita",
"Linda Erlina",
"Khaerunissa Anbar Istiadi",
"Fadilah Fadilah",
"Retno Wahyuningsih",
"Anna Rozaliyani",
"Ayu Eka Fatril",
"Rafika Indah Paramita",
"Linda Erlina",
"Khaerunissa Anbar Istiadi"
],
"abstract": "Candida krusei is a Candida non-albicans species with a high prevalence, which causes candidaemia. Current treatment guidelines include fluconazole as a primary therapeutic option for the treatment of these infections; however, it is only a fungistatic against Candida spp., and both inherent and acquired resistance to fluconazole have been reported. C. krusei species is also reported as the only Candida sp. which has an intrinsic resistance factor to fluconazole. Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei. The purpose of this study was to investigate the genome of clinical C. krusei isolates and correlate the resistant phenotypes with mutations in resistance genes. A total of 16 samples of C. krusei from clinical samples from hospitals in Jakarta were used in the experiment. All colonies were extracted using the QIAamp DNA Mini Kit. The library was prepared using the Illumina DNA Prep Kit. The sequencing process was carried out on the Illumina MiSeq Platform using a 2x301 paired-end configuration. FASTQ raw files are available under the BioProject Accession Number PRJNA819536 and Sequence Read Archive Accession Numbers SRR18739949 and SRR18739964.",
"keywords": [
"Candida krusei",
"fluconazole",
"genomic profiling",
"resistance gene"
],
"content": "Introduction\n\nCandida krusei (teleomorph Pichia kudriavzevii) is an opportunistic fungal pathogen.1 It is the fourth most common non-albicans species (NAC) species causing invasive candidiasis and candidemia.2,3 C. krusei is among unique species with natural resistance towards fluconazole, a commonly used antifungal for Candida infections.4 Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei. The purpose of this study was to investigate the genome of clinical isolates of C. krusei and correlate the resistant phenotypes with mutations in resistance genes.\n\n\nMethods\n\nThe C. krusei clinical sample were retrieved from the Department of Parasitology Culture Collection, Faculty of Medicine, Universitas Indonesia. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, catalog number: 51304) with bead-beating methods.5 100 ÎŒL culture of C. krusei were added into 1.5 ml microcentrifuge tube containing 200 ÎŒL ATL buffer (Qiagen). Three spoon full of sterilized sand were added into the mixture for bead-beating, which was performed for 10 minutes. 20 ÎŒL Proteinase K (Qiagen) were added and the mixture was incubated at 56°C until completely lysed (three hours), and agitated every 10 minutes during incubation time, until homogeneous. 200 ÎŒL Buffer AL (Qiagen) were added and the mixture was incubated at 70°C for 10 minutes. 200 ÎŒL pure ethanol were added and the mixture was transferred into the QIAamp Mini spin column (Qiagen). Column were centrifugated at 6000 à g for one minute and the flow-through were discarded. QIAamp Mini spin columns were placed innew 2 mL collection tubes and 500 ÎŒL Buffer AW1 were added into the column. Columns were centrifugated at 6000 à g for one minute and the flow-through was discarded. QIAamp Mini spin columns were placed into new 2 mL collection tubes and 500 ÎŒL Buffer AW2 (Qiagen) were added. Columns were centrifugated at 17000 à g for three minutes and the flow-through was discarded. QIAamp Mini spin columns were transferred into new 1.5 mL microcentrifuge tubes, and 200 ÎŒl distilled water were added. Columns were incubated at room temperature for one minute and centrifugated at 6000 à g for one minute to elute the DNA. The quality of extracted DNA (A260/280) was measured using a spectrophotometer (NanoDrop ND-1000). The quantity of extracted DNA was measured with Qubit 4.0 Fluorometer using the dsDNA HS Assay kit.\n\nDNA libraries were prepared using the Illumina DNA Preparation Kit. The index used for the library preparation was Integrated DNA Technologies, Inc (IDT) for Illumina Nextera Indexes for ligation step. The library construction steps were as follows:\n\nTagmentation\n\n100 ng DNA were added into a 96-well plate and mixed with tagmentation master mix from Illumina Nextera Kit (Illumina). The mix were then incubated at 55°C in a thermal cycler (The Applied Biosystems ProFlex PCR System) for 15 minutes.\n\nPost tagmentation clean-up and amplification of tagmented DNA\n\nIllumina Nextera Kit tagment stop buffer (Illumina) were added to the tagmentation reaction and incubated at 37°C for 15 minutes. The mixture was washed with Illumina Nextera Tagment wash buffer (Illumina) on the magnetic stand. The tagment Wash Buffer was discarded and Nextera PCR master mixes (Illumina) were added onto the beads. Index adapters were added as sample barcoding. The mixture was amplified in thermal cycler.\n\nLibraries clean-up\n\nThe beads were added into the supernatant of the mixture and washed twice using 80% ethanol on a magnetic stand. Nextera resuspension buffer (Illumina) reagents were added onto beads and final libraries were retrieved from the supernatant. The libraries of each sample were pooled.6\n\nThe barcoded DNA libraries were sequenced using an Illumina Miseq Platform on the v3-Flow Cell. The DNA library was denatured according to the manufacturerâs protocol.7 For quality control, the library was spiked with 1% PhiX Control v3. The sequencing run produced 2 à 301 bp paired-end libraries. The data were deposited to the NCBI Sequence Read Archive (SRA) under BioProject. Total raw reads were obtained using FastQC software, and the total raw bases and percentage of Q30 were evaluated using q30 python scripts.8\n\nThis research was approved by the Faculty of Medicine Universitas Indonesia Ethical Committee (approval number: 970/UN2.F1/ETIK/PPM.00.02/2021).\n\n\nResults\n\nThe whole raw genome sequence data of 16 clinical isolates of C. krusei from the Department of Parasitology, Faculty of Medicine of Universitas Indonesia were deposited into NCBI data under BioProject accession number PRJNA819536 and SRA SRR18739949-SRR18739964. The DNA quality and quantity of the samples are shown in Table 1. Information regarding the raw data is described in Table 2. These raw data of C. krusei genome are useful for genome profiling and correlating the resistant phenotypes with mutations in resistance genes.\n\n\nData availability\n\nRaw data (FASTQ) files have been deposited into National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov).\n\nBioProject: Raw sequence reads of Candida krusei from clinical samples, Accession Number: PRJNA819536; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA819536\n\nBioSample: Pathogen: clinical or host-associated sample from Pichia kudriavzevii, Accession Number: SAMN26901813; https://identifiers.org/ncbiprotein:SAMN26901813\n\nBioSample: Pathogen: clinical or host-associated sample from Pichia kudriavzevii, Accession Number: SAMN26901828; https://identifiers.org/ncbiprotein:SAMN26901828\n\nRaw sequence reads of Candida krusei from clinical samples:\n\nSequence Read Archive: Raw sequence reads of Candida krusei from clinical samples, Accession Number: SRR18739949; https://identifiers.org/insdc.sra:SRR18739949\n\nSequence Read Archive: Raw sequence reads of Candida krusei from clinical samples, Accession Number: SRR18739964; https://identifiers.org/insdc.sra:SRR18739964",
"appendix": "References\n\nCooper CR: Yeasts pathogenic to humans. Elsevier B.V;2011; vol. 1.\n\nPfaller MA, Jones RN, Castanheira M: Regional data analysis of Candida non- albicans strains collected in United States medical sites over a 6-year period, 2006-2011. Mycoses. Oct. 2014; 57(10): 602â611. PubMed Abstract | Publisher Full Text\n\nLamping E, et al.: Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei. Antimicrob. Agents Chemother. Feb. 2009; 53(2): 354â369. PubMed Abstract | Publisher Full Text\n\nGuinea J, Sánchez-Somolinos M, Cuevas O, et al.: Fluconazole resistance mechanisms in Candida krusei: the contribution of efflux-pumps. Med. Mycol. Sep. 2006; 44(6): 575â578. PubMed Abstract | Publisher Full Text\n\nScharf S, Bartels A, Kondakci M, et al.: Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens. Int. J. Med. Microbiol. Sep. 2020; 310(6): 151443. PubMed Abstract | Publisher Full Text\n\nIllumina: Illumina DNA Prep Reference Guide.2020.Reference Source\n\nIllumina: MiSeq System Denature and DIlute Librariers Guide.2018.Reference Source\n\nChen S: q30 python script.2016.Reference Source"
}
|
[
{
"id": "147383",
"date": "30 Aug 2022",
"name": "Xuepeng Sun",
"expertise": [
"Reviewer Expertise Genomics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDrug resistance is a serious problem for human disease. As the human pathogen C. krusei has natural resistance to the commonly used azole fungicides, it is of particular importance to explore the molecular basis underlying the resistance. This study provides high-depth sequence data for 16 clinic isolates of C. krusei, which in combination with published C. krusei genome will provides new information for correlating the mutations with the resistance phenotypes. Due to the potential interest of the data to scientific community and the quality of the manuscript, I would suggest an acceptance of this manuscript in F1000Reseach.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
},
{
"id": "159413",
"date": "09 Jan 2023",
"name": "Atanu Banerjee",
"expertise": [
"Reviewer Expertise Mycology. Antifungal resistance",
"Candida species",
"Efflux pumps",
"structure-function studies",
"sequence",
"and structural bioinformatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Fadilah et al, presents the whole genome sequence data of clinical isolates of C. krusei. While the genome sequence data generated could be useful to advance our knowledge regarding factors that might be responsible for the intrinsic azole resistance exhibited quite often by C. krusei isolates, there are some significant gaps that need to be filled to make the manuscript more useful for readers. I elaborate on some of them:\nThe introduction seems extremely brief and does not bring the importance of the work into the proper context. For instance, the authors write \" Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei\" and then directly mention \"The purpose of this study was to investigate the genome of clinical isolates of C. krusei and correlate the resistant phenotypes with mutations in resistance genes.\". The two statements do not bring the perspective and importance into place appropriately.\n\nAlso, there isn't much background literature provided in this section.\n\nThe authors mention non-albicans Candida (NAC) species incorrectly in the abstract and introduction. It should be corrected.\n\nThe authors do not provide any background information related to the clinical isolates, except the center. It is extremely important to at least briefly provide some background information on the isolates.\n\nWhile the authors state that \"The purpose of this study was to investigate the genome of clinical isolates of C. krusei and correlate the resistant phenotypes with mutations in resistance genes\", there is no information provided regarding the same. It limits the importance of the paper.\n\nIn the keywords, \"Candida krusei\" is non-italicized. \"Resistance gene\" does not make much sense and should be replaced with something more meaningful. The same should also be replaced elsewhere in the MS.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-593
|
https://f1000research.com/articles/12-347/v1
|
29 Mar 23
|
{
"type": "Research Article",
"title": "Assessing structure and disorder prediction tools for de novo emerged proteins in the age of machine learning",
"authors": [
"Margaux Aubel",
"Lars Eicholt",
"Erich Bornberg-Bauer",
"Margaux Aubel",
"Lars Eicholt"
],
"abstract": "Background: De novo protein coding genes emerge from scratch in the non-coding regions of the genome and have, per definition, no homology to other genes. Therefore, their encoded de novo proteins belong to the so-called \"dark protein space\". So far, only four de novo protein structures have been experimentally approximated. Low homology, presumed high disorder and limited structures result in low confidence structural predictions for de novo proteins in most cases. Here, we look at the most widely used structure and disorder predictors and assess their applicability for de novo emerged proteins. Since AlphaFold2 is based on the generation of multiple sequence alignments and was trained on solved structures of largely conserved and globular proteins, its performance on de novo proteins remains unknown. More recently, natural language models of proteins have been used for alignment-free structure predictions, potentially making them more suitable for de novo proteins than AlphaFold2. Methods: We applied different disorder predictors (IUPred3 short/long, flDPnn) and structure predictors, AlphaFold2 on the one hand and language-based models (Omegafold, ESMfold, RGN2) on the other hand, to four de novo proteins with experimental evidence on structure. We compared the resulting predictions between the different predictors as well as to the existing experimental evidence. Results: Results from IUPred, the most widely used disorder predictor, depend heavily on the choice of parameters and differ significantly from flDPnn which has been found to outperform most other predictors in a comparative assessment study recently. Similarly, different structure predictors yielded varying results and confidence scores for de novo proteins. Conclusions: We suggest that, while in some cases protein language model based approaches might be more accurate than AlphaFold2, the structure prediction of de novo emerged proteins remains a difficult task for any predictor, be it disorder or structure.",
"keywords": [
"de novo proteins",
"disorder",
"pLDDT",
"protein structure",
"structure predictions",
"AlphaFold",
"Natural language models"
],
"content": "Introduction\n\nThe existence of proteins arising from non-coding parts of the genome, also known as de novo emergence, was once considered almost impossible [Zuckerkandl, 1975, Jacob, 1977]. However, with the completion of the yeast genome project [Dujon, 1996] and the discovery of âorphansâ, which are defined as proteins lacking any detectable homology to proteins in sister species, the concept of de novo protein emergence called for a reevaluation. Among orphan proteins, de novo proteins are unique, as they can be shown using further methods including synteny analysis, to have been born from formerly non-coding DNA [Vakirlis et al., 2020]. Accordingly, their sequence composition might resemble proteins with random sequences, yet to an unknown degree [Oss and Carvunis, 2019, Bornberg-Bauer et al., 2021]. In particular, in several studies de novo proteins have been predicted to be highly disordered which can at least partially be attributed to their high GC (guanine-cytosine) content [Wilson et al., 2017, Landry et al., 2015, Basile et al., 2017]. Other studies reported de novo proteins to contain a lower proportion of structural disorder [Xie et al., 2019] than conserved proteins, while yet other studies report no significant difference in predicted disorder between de novo and conserved proteins [Schmitz et al., 2018, Dowling et al., 2020]. The differences between these findings may be caused by the usage of different species and age groups studied in the datasets, but also to different methods used.\n\nExperimental structure determination of de novo proteins is still in its infancy, due to difficulties in purification and methodological limitations [Eicholt et al., 2022]. Therefore, several studies include computational structure [Vakirlis et al., 2020; Lange et al., 2021] and disorder predictions [Schmitz et al., 2018, Xie et al., 2019]. However, most structure predictors are based on multiple sequence alignments (MSA) and training sets containing only structures of conserved proteins [Jumper et al., 2021, ErdÅs et al., 2021, Hu et al., 2021]. While these methods certainly provide information on a wide array of protein properties, it is not yet clear how reliable these methods are for proteins with no detectable homology to known proteins, such as de novo proteins, but also random sequence proteins or de novo designed proteins [AlQuraishi, 2021]. A more reliable option for predicting de novo protein structures could be programs based on protein language models (pLM) since these models do not require any MSA [Michaud et al., 2022]. Instead, pLMs have learned general sequence architectures in proteins and how these relate to structures or structural elements. Using the language analogy, this is similar to learning grammar and building whole sentences from single words and words from letters [Michaud et al., 2022, Chowdhury et al., 2022a, Wu et al., 2022a, Lin et al., 2022a]. While structural properties have been computationally analysed for several sets of de novo proteins [Dowling et al., 2020, Heames et al., 2020, Wilson et al., 2017, Carvunis et al., 2012], only four de novo protein structures have been experimentally characterised [Lange et al., 2021, Bungard et al., 2017, Matsuo et al., 2021; Her et al., 2019]. To date, no confirmed de novo protein structure has been completely solved experimentally.\n\nAFGP is a de novo emerged antifreeze glycoprotein family in Arctic codfish [Baalsrud et al., 2018]. The family emerged de novo in the arctic codfish lineage around 15 mya (million years ago) with extant protein variants existing in several arctic codfish [Baalsrud et al., 2018]. AFGP enables arctic codfish to survive the subzero temperatures of their biotope by preventing accumulation of ice crystals in the blood [Cheng, 1998]. The secretion of AFGP into the blood is induced by a signal peptide, that is followed in the sequence by a post-translationally removed short glutamine-rich region, and T-(A/P)-A repeats up to 200 amino acids long [Zhuang et al., 2019]. The Threonine residues of the repeats are glycolysated and bind to the surface of emerging ice crystals. AFGP blocks thereby the addition of water molecules to the ice crystal and decreases the freezing point of the blood serum. [Cheng, 1998, Devries, 1971]. Nuclear magnetic resonance (NMR) spectroscopy of de novo emerged AFGP from Boreogadus saida revealed that AFGP is a highly dynamic and mostly disordered protein that can form polyproline II helices [Her et al., 2019]. The AFGP in Antarctic notothenioid fish, while not emerged de novo but from a trypsinogen-like serine protease gene [Cheng, 1998, Chen et al., 1997, Weisman, 2022], exhibits similar dynamic behaviour of the convergently emerged repetitive region [Giubertoni et al., 2019].\n\nBsc4 is a de novo gene specific to Bakerâs yeast Saccharomyces cerevisae with a transcribed locus in homologous species, while lacking an open reading frame (ORF) in all transcripts except in S. cerevisiae [Cai et al., 2008]. Protein expression of Bsc4 is upregulated during the stationary growth phase of Bakerâs yeast. The deletion of Bsc4 is lethal when combined with the deletion of the conserved genes RPN4 and DUN1, but not contrariwise. RPN4 and DUN1 play both a role in DNA repair pathways [Cai et al., 2008, Pan et al., 2006, Li et al., 2010]. This could indicate an important role of Bsc4 in the DNA repair pathway of yeast. The 132 amino acid long protein was analysed using tryptophan fluorescence and near-ultra violet (UV) circular dichroism (CD) and is considered to be of a molten globule structure consisting of abundant β-sheets while lacking tight packaging. According to ion mobility-mass spectrometry, Bsc4 can build homopolymer assemblies up to hexamers [Bungard et al., 2017].\n\nThe role of the putative de novo protein Goddard was detected using fertility screens in Drosophila melanogaster [Gubala et al., 2017]. Null-alleles of endogenous Goddard render male D. melanogaster infertile while not affecting viability. Using a combination of antibody staining and confocal microscopy, Goddard was found to localise to elongated sperm axonemes. The absence of Goddard results in failed individualisation of spermatids and therefore causes sterility in male fruit flies. The structure of Goddard was analysed using a combination of CD, thermal shift assay (TSA), NMR and ab initio structure prediction followed by molecular dynamics simulations [Lange et al., 2021]. All methods indicate a central α-helix but high amounts of disorder in the rest of the Goddard protein. The central α-helix is conserved in Goddard orthologs and has been retained in the structure for at least 50 my, according to ancestral sequence reconstruction [Lange et al., 2021].\n\nThe human specific de novo protein NCYM is the cis-antisense transcript of the MYCN oncogene. Both genic sequences are overlapping, but their coding regions do not overlap [Weisman, 2022, Suenaga et al., 2020]. NCYM was the first de novo gene whose role in cancer progression was detected in vivo and has been structurally analysed [Matsuo et al., 2021]. The SUMO-tagged NCYM protein was subjected to vacuum-UV CD and measurements were evaluated using an early neural network [Matsuo et al., 2008]. The neural network subtracted the structural content of the SUMO-tag, thereby elegantly bypassing the cleavage of the tag from the 109 amino acid long NCYM [Matsuo et al., 2021, 2008]. According to the predictions enhanced with CD data, NCYM is mostly disordered but contains several stretches of α-helices and some smaller β-sheets [Matsuo et al., 2021].\n\nThe structure-function paradigm suggests that a protein needs a defined structure to be functional [Wright and Dyson, 1999]. However, research on disordered proteins demonstrated that this paradigm does not always hold up and that disordered proteins can carry out important biological functions too [Uversky and Dunker, 2010]. For example, many binding motifs are located in disordered protein regions and disordered proteins are known to be involved in signalling pathways [Ali et al., 2020]. However, it is widely asserted that a defined tertiary structure is complex and presumably difficult to attain from scratch, i.e., without adaptation. Therefore, de novo proteins are often assumed to contain little structural content [Bungard et al., 2017, Wilson et al., 2017, Schmitz et al., 2018]. Many de novo protein studies have included disorder predictions in their analyses [Schmitz et al., 2018, Xie et al., 2019, Carvunis et al., 2012, Wilson et al., 2017]. During protein evolution, such a lack of well-defined structure might even be an advantage for newly emerging proteins under some circumstances. Indeed, highly disordered proteins were shown to be soluble and less prone to aggregation [Linding et al., 2004], which has been described as a favoured trait in protein evolution. Since solubility is required for most protein functions a majority of protein sequences have evolved towards lower aggregation propensities [Monti et al., 2021].\n\nThe amount of disorder of a protein is relatively straightforward to predict from its amino acid sequence. Several algorithms are available as online interfaces or local programs [Dosztányi et al., 2005, ErdÅs et al., 2021, Necci et al., 2021, Hu et al., 2021, Hanson et al., 2019]. IUPred is among the most frequently used disorder predictors, especially in de novo protein studies [Schmitz et al., 2018, Wilson et al., 2017, Xie et al., 2019, ErdÅs et al., 2021]. IUPred is not based on evolutionary information but physical properties of the amino acids to be structure or disorder promoting, by using energy estimations of the single amino acids in the sequence [ErdÅs et al., 2021, Dosztányi et al., 2005]. These energy estimations are derived from known contacts between amino acids in experimentally determined structures of globular proteins. This results in a 20x20 matrix containing energy estimations for each pair of amino acids. The final disorder probability for each residue depends on the energy estimation of the specific amino acid and its neighbouring residues. Accordingly, IUPred appears to be most suitable for proteins without known homologs.\n\nRecently, the final results of Critical Assessment of protein Intrinsic Disorder prediction (CAID) [Necci et al., 2021], demonstrated that there are many precise machine learning-based disorder predictors available that outperform IUPred in accuracy [Hanson et al., 2019, Hu et al., 2021]. However, most of the top disorder predictors rely on evolutionary information, which may not be ideal for prediction of de novo proteins and other unusual sequences. flDPnn is among the few top disorder predictors that do not rely on evolutionary information, making it a promising predictor for de novo proteins. The true positive rate of predicted disorder is highest for flDPnn [Hu et al., 2021] when compared to the other predictors (SPOT, IUPred âlongâ and âshortâ).\n\nStructural biology has changed with the advent of DeepMindâs AlphaFold2 (AF2) [Jumper et al., 2021] and structure predictors gained ground for many different research areas [Lupas et al., 2021; Marx, 2022]. As of now, the AF2 protein structure database, a joint project of DeepMind and EMBL-EBI, contains more than 100 million high quality predicted protein structures, e.g. from Homo sapiens & D. melangoaster [Varadi et al., 2021]. The abundant high-quality predictions in the AF2 PDB have already been leveraged for improved geometric pre-training of structure predictors of the next generation [Zhang et al., 2022]. Until yet training was only limited to experimentally solved structures [Zhang et al., 2022]. Novel structure predictors such as AF2 are particularly promising for studying de novo proteins due to the aforementioned lack of experimentally determined structures. However, AF2 has its own limitations. The properties of de novo proteins such as high disorder, short length and lack of homologous proteins make structure prediction of those de novo proteins a challenging task for AF2. Accordingly, results must be interpreted with caution [Monzon et al., 2022]. The lack of homologous sequences in particular might pose a problem for AF2 since it is based on co-evolutionary data extracted from MSAs. AF2 uses correlations of co-occurences between amino acids in an MSA to deduce the proximity of those amino acids in the protein structure [Jumper et al., 2021; Michaud et al., 2022]. De novo proteins do not necessarily lack homology entirely, since they can also appear in a whole lineage, as in the case for AFGP. In those cases, an MSA could provide co-evolutionary data to predict secondary structure elements but likely not for the abundant disordered regions in which the assumption of positional homology could be violated [Lindorff-Larsen and Kragelund, 2021]. Disordered regions are highly flexible in space, while predictions based on MSAs assume that the amino acid position in a sequence correlates to a fixed position in the structure [Lindorff-Larsen and Kragelund, 2021]. Nevertheless, de novo proteins are assumed to be mutationally remote in sequence space (and therefore evolutionary unrelated) to areas of well characterised protein families in structural space. Therefore, recent structure prediction programs based on protein language models (pLMs) could yield more realistic results for de novo protein structure since they are alignment-free.\n\nWe will summarise existing structural evidence for different de novo proteins and methodological limitations, with a focus on the most widely used disorder and structure predictors, IUPred3 and AlphaFold2, respectively. A major caveat for disorder comparison is that computational predictions of de novo protein properties are difficult to compare between studies, because of differences in parameters used [Schmitz et al., 2018]. Here, we used four experimentally characterised, or rather approximated, de novo proteins to illustrate that results of different prediction algorithms differ significantly in most cases, and do not always align well with the experimental evidence at hand. Finally, we will focus on longer standing questions on structure predictions of de novo proteins and on novel questions that were raised with the advancement of machine learning (ML) based structure predictions: Specifically we ask, how reliable structure predictions for de novo proteins are and what possible pitfalls during analysis of those predictions may arise. Enabled by the advancement of the structure prediction field, the structural analysis of de novo proteins will thus bring more light into the âdark protein spaceâ, the hitherto non-characterised region of sequence space. Therefore, novel structures and folds could be discovered and provide new starting points for protein engineering and deeper insights on protein evolution.\n\n\nMethods\n\nOnly de novo protein sequences with experimental evidence on structure were taken for analyses. For this purpose, available peer-reviewed publications on de novo emerged proteins were manually screened for candidates [Weisman, 2022, Bornberg-Bauer et al., 2021]. After screening literature for appropriate candidates and removing i) de novo proteins without structural information and ii) falsely identified de novo emerged candidates, the de novo protein sequences were downloaded from UniProt (RRID:SCR_002380), accessed in December 2022. The UniProt accession numbers can be found in Table 1 and all sequences used are included in the underlying data as fasta files. Eight conserved proteins with experimentally determined structures containing different amounts of disorder, four with low and four with relatively high amounts of disorder were taken as controls. The observed values for the fraction of residues in disordered regions were taken from MobiDB (RRID:SCR_014542). For accession numbers and species of origin, see Table 1. Amino acids were counted with a custom Python 3.10 (RRID:SCR_008394) script available on Zenodo: https://doi.org/10.5281/zenodo.7615407 and zivgitlab/l_eich04/structure_predictions_de_novo.\n\nDisorder predictions were performed locally using IUPred3 [ErdÅs et al., 2021] (RRID:SCR_014632), using the parameters âshortâ and âlongâ predictions and flDPnn [Hu et al., 2021] using default parameters. The fraction of residues in a disordered region (referred to simply as fraction from hereon) was determined by calculating the average of the binary predictions for disorder in flDPnn. For IUPred, the binary predictions were calculated first by assigning the value 1 if predicted disorder was >0.5, and 0 if predicted disorder was <0.5 and then averaged to get the fraction of residues in disordered regions. Statistical analysis and plots were done in RStudio 4.2.2. (RRID:SCR_000432) [RStudio Team, 2020, R Core Team, 2022]. To determine whether the observed differences were significant (p-value < 0.05), the Kruskal-Wallis rank sum test followed by the Dunn test were performed and p-values adjusted using Holm method from FSA package [Ogle et al., 2022]. Plots were generated using the ggplot2 package [Wickham, 2016]. The code used in R is available as âR_stats_plots.txtâ on Zenodo: https://doi.org/10.5281/zenodo.7615407 and on zivgitlab/l_eich04/structure_predictions_de_novo. All software tools used are freely available.\n\nStructural predictions were performed using AlphaFold v2.1.1 on High Performance Computing Cluster PALMA II (University of Muenster). RGN2 (Number of recycles 1), OmegaFold (Number of cycles 4) and ESMfold (Number of cycles 3) predictions were performed using respective Google Colabs (RRID:SCR_018009) [Chowdhury et al., 2022b, Wu et al., 2022b, Lin et al., 2022b]. For each the standard number of cycles/recycles were chosen. Predictions with the highest mean pLDDT were selected. The pLDDT of different segments were examined with ChimeraX 1.5 [Pettersen et al., 2021] (RRID:SCR_015872) and the commandâcolor bfactor palette alphafoldâ. PyMOL 2.5.2. [Schrödinger, LLC, 2015] (RRID:SCR_000305) was used for structural alignments and visualizations. AlphaPickle [Arnold, 2021] was used to pull pLDDT values for each residue from the b factor column of PDB files. Two N-terminal residues were removed for predictions of AlphaFold2, Omegafold and ESMfold since RGN2 predictions exclude the last two N-terminal residues [Floristean, 2022]. Violin plots were created using Python 3.10 (RRID:SCR_008394) with libraries matplotlib [Hunter, 2007] (RRID:SCR_008624) and pandas [Wes McKinney, 2010] (RRID:SCR_018214). Kruskal-Wallis rank sum test and Dunn test were performed and p-values adjusted using Holm method in RStudio [RStudio Team, 2020] as described for the disorder predictions. AlphaFold2 predictions of AFGP polyprotein (O13083) and Antifreeze glycopeptide (Q90401) of Dissostichus mawsoni (Antarctic cod) were not performed but downloaded from AlphaFold Protein Structure Database.\n\nAll software tools used are freely available. All code and original result files are available in the extended data on Zenodo: https://doi.org/10.5281/zenodo.7615407. Code is additionally available on zivgitlab: https://zivgitlab.uni-muenster.de/l_eich04/structure_predictions_de_novo.\n\n\nResults\n\nHere, we compare the performance of flDPnn [Hu et al., 2021], which performs best according to CAID, to the latest version of IUPred [ErdÅs et al., 2021], the most widely used predictor. We focus on de novo proteins that were experimentally characterised, namely AFGP, Bsc4, Goddard and NCYM (see Figure 1). According to experimental evidence, all four de novo proteins contain disordered regions. When predicting the disorder of AFGP with flDPnn as described in the methods, around 80 % of residues are predicted to be disordered. IUPred âlongâ (IUPredL) predicts around 70 % and IUPred âshortâ (IUPredS) only 25 %. Here, the biggest and most significant difference can be observed between the predictors, with all p-values < 0.005. This can be partly attributed to AFGP being by far the longest protein used in the analyses with 700 amino acids (see Table 1). Bsc4 predictions are highly similar between the three predictors and indicate low amounts of disorder. The median value is around 0.1 for all predictors while the fraction and mean show more variation from 0-13 % and 0.12-0.19 respectively. All predictors that were used on the Goddard sequence here result in high disorder with around 75 % of all residues in a disordered region and a mean score of about 0.7 for Goddard predictions. Predictions of Goddard differ significantly between IUPredS and IUPredL (p-value = 0.0157), highlighting the importance of the right choice of parameters. NCYM disorder predictions recognise 10 % of residues as disordered with mean probability for disorder of around 0.25. There is no significant difference between the disorder predictors, which can be partially attributed to the short length of the protein (109 amino acids).\n\nMean values are displayed as diamond shapes, median as lines and crosses display the fraction of disordered residues. Significant differences between the disorder predictors are indicated by stars (***<0.0005; **<0.005; *<0.05).\n\nProbabilities for disorder in all de novo protein sequences, except NCYM, differ significantly between the predictors with p-values below 0.05 as shown in Figure 1 and underlying data. Most importantly, the fraction of disordered residues varies greatly depending on the predictor that was used here.\n\nAs shown in Figure 1 the difference between fraction of disordered residues and mean probability for disorder over all residues in the sequence can deviate significantly. While both values are indicators for disorder in a protein, they have slightly different implications. On the one hand, when only the average probability for disorder of all residues is reported, little information on actual amount of disorder in a protein is gained. It is impossible to distinguish between a theoretical protein with some highly disordered (probability close to 1) and some highly structured regions (probability close to 0) and a protein with ambiguous probability for disorder (probability around 0.5) in the whole sequence. Looking at the predictions performed with the de novo proteins, flDPnn predicts very similar average probabilities for disorder in AFGP and Goddard (0.57 and 0.61). The fraction of residues in a disordered region not only differs more between the two than the average disorder, but the trend is actually reversed (82 and 75 %). Goddard is predicted to contain 75 % of disordered residues and AFGP 82 %. On the other hand, when using the fraction of residues that are predicted to be disordered in the protein, the minor differences in probabilities for disorder disappear. For example, a theoretical protein that is just below the threshold for a disordered region of 0.5 in the majority of the sequence, is indistinguishable from a protein with a probability for disorder of 0 across the whole sequence. The de novo proteins NCYM and Bsc4 predicted here have a very similar fraction of disordered residues, but judging from the average probability they seem to differ much more.\n\nFor a more general comparison, we took eight conserved and experimentally solved structures and applied the same prediction algorithms. All eight proteins have varying amounts of disorder based on the PDB structures which we collected from the disorder database MobiDB. The observed disorder is indicated in Figure 2 and is compared to the values predicted by the three programs that were also applied to the de novo proteins before (IUPredL, IUPredS and flDPnn). Four of the proteins contain low amounts of disorder below 25 % according to the experimentally determined structures. Results of all three disorder predictors are close to the observed values. The four proteins that contain higher amounts of disorder (p53, Nop10, AF9 and alpha-synuclein), vary much more between the predictors and have higher amounts of observed disorder than is predicted.\n\nThe disorder for four highly structured and four highly disordered proteins with experimentally resolved structures was predicted with IUPredS, IUPredL and flDPnn. The proteins are ordered from low disorder on the left to high disorder on the right. While flDPnn predicts the disorder for all lower than observed values, the trend remains the same. IUPredL and IUPredS are closer to the observed values for structured proteins, but deviate from the trend for disordered proteins.\n\nHere, we will present the structure predictions of de novo proteins AFGP from B. saida, Bsc4, Goddard and NCYM [Her et al., 2019, Bungard et al., 2017, Lange et al., 2021, Matsuo et al., 2021] with AF2, OF, RGN2 and ESMFold [Jumper et al., 2021, Wu et al., 2022a, Chowdhury et al., 2022a, Lin et al., 2022a]. As mentioned before, there is no experimentally determined structure of a de novo evolved protein that can serve as ground truth when comparing prediction programs. All programs provide a predicted local distance difference test (pLDDT) [Jumper et al., 2021, Mariani et al., 2013] based on the AF2 structure module to evaluate the prediction confidence of each residue of the model.\n\nIt is important to note here that pLDDT is a confidence measure of each program for the predictions performed by itself and not to compare the confidence of predictions of different programs to each other. In the following, when we compare the pLDDTs of different programs we are thereby not assessing which program provides the most reliable prediction. Also, low pLDDT can be an indicator of high disorder [Akdel et al., 2022, Ruff and Pappu, 2021].\n\nAdditionally as controls, we performed structure predictions in the same manner as for the de novo proteins, for evolutionary conserved and both structurally solved and experimentally confirmed intrinsically disordered proteins (IDPs); p53, Nop10, AF-9 and Alpha-synuclein (Figure 4 & Table 1).\n\nThe pLDDT values for the predictions of AFGP from B. saida (Figure 3A) are significantly different from each otherâs prediction. We found that all programs predict an N-terminal α-helix while the rest of the structure is ribbon-like, indicative of disorder. Only ESMfold predicts three additional shorter helices (T222-T225, T414-A418, T672-A680). The predictions of AFGP show differing pLDDT between predictors while all predictions effectively display high levels of disorder. The pLDDT values of Bsc4 are more similar to each other except for predictions obtained from OF and ESMfold. These two differ significantly in pLDDT from those obtained with RGN2 and different secondary elements are predicted (Figure 3B). AF2 predicts smaller β-sheets and RGN2 does not predict any β-sheets. However, in a lower pLDDT-ranked AF2 structure (see extended data), the determined β-sheets are similar predicted and almost identical to those by OF and ESMfold. Predictions of Bsc4 have similar pLDDT values while the underlying predicted structures are not. Goddard is structurally composed of a confirmed central α-helix and disordered termini [Lange et al., 2021]. The predicted structures of Goddard are similar by eye (Figure 3C) and if the models are structurally aligned to the central α-helix of the AF2 model as a target this similarity becomes even more apparent (Figure 3C, Cα-RMSD = 0.770Ã
). AF2, OF and RGN2 predict the majority of the helix with very high confidence which decreases only towards the termini when employing RGN2 and OF. ESMfold predicts the structure of Goddard only with low to very low confidence, with significant difference in pLDDT to other predictors.\n\nA: AFGP, to α-helix (M1-A28), Cα-RMSD = 0.663Ã
. B: Bsc4, to α-helix (K62-R83), Cα-RMSD = 1.603Ã
. C: Goddard, to α-helix (S38-I80), Cα-RMSD = 0.770Ã
. D: NCYM, to α-helix (N50-E66), Cα-RMSD = 5.278Ã
. Significant difference in pLDDT is indicated by *** (p-value < 0.0005).\n\nFor NCYM (Figure 3D) all programs except OF predict one α-helix, while OF predicts two. The α-helix predicted by OF and AF2 is longer than the one predicted by RGN2. OF predicts an additional α-helix. RGN2 is the only method that predicts β-sheets (R60-C64, C104-I107). These β-sheets overlap with the α-helices predicted by the other programs, explaining the lower RMSD (Cα-RMSD = 5.278Ã
). In this case, the pLDDT of AF2 is higher than the pLDDT of the other programs, which have comparably low values for predictions of NCYM.\n\nThe question remained, if both the overall results of structure predictions of de novo proteins and their variations are a consequence of i) disorder level and/or of ii) lack of sequence homology. Therefore, we performed structure predictions of p53, Nop10, AF9 and alpha-synuclein with the same tools as before (AF2, OF, ESMfold, RGN2). These control proteins are all evolutionary conserved and are experimentally confirmed intrinsically disordered proteins (IDPs). For each of the four control IDPs the predicted secondary structure elements are approximately in the same position for all prediction tools. Only lengths of secondary elements and of ribbon-like structures, indicating disorder, are variying between each prediction (Figure 4). The respective predictions of all four control IDPs show broadly significant differences in pLDDT but not in all cases (Figure 4). The number of significant differences in pLDDT for predictions of IDPs is lower than for de novo protein predictions (Figures 3 and 4). In general the pLDDT values for the predictions of experimentally conserved IDPs are higher than for de novo proteins (Table 2).\n\nA: p53, to β-sheets (C123-E203), Cα-RMSD = 19.198Ã
. B: Nop10, to β-sheets (M4-T16), Cα-RMSD = 0.269Ã
. C: AF9, to to α-helices (K500-S566), Cα-RMSD = 1.558Ã
. D: alphasynuclein, to α-helix (M1-G40), Cα-RMSD = 1.140Ã
. Significant difference in pLDDT is indicated by *, **, *** (p-value < 0.05 < 0.005 < 0.0005).\n\n\nDiscussion\n\nResearch on functional disordered proteins is increasing and so is the need to structurally characterise and detect disordered protein regions [Alderson et al., 2022, Lindorff-Larsen and Kragelund, 2021, Ali et al., 2020, Bruley et al., 2022]. For newly detected but also newly emerged proteins, as de novo proteins often are, disorder is an interesting hallmark to investigate because disorder promotes high solubility, disfavours aggregation [Linding et al., 2004, Monti et al., 2021], and at the same time, is often associated with a high density of binding motifs which make the protein amenable to many regulatory processes [Ali et al., 2020]. Disorder predictions are therefore often used to gather information about de novo proteins by comparing them to: i) conserved proteins [Xie et al., 2019, Carvunis et al., 2012], ii) different age groups in de novo proteins [Schmitz et al., 2018, Wilson et al., 2017, Carvunis et al., 2012, Dowling et al., 2020] and iii) random sequence proteins [Heames et al., 2022]. With many studies relying on the disorder predictions of de novo proteins and only few attempts to experimentally characterise their disorder [Heames et al., 2022, Bungard et al., 2017], it is paramount that the predictors used are precise enough for de novo protein sequences to allow for the conclusions drawn. Further, to compare predictions not just in single studies but more easily between different studies, consensus about prediction methods and parameters is needed.\n\nComparing disorder predictions of the four de novo proteins with each other, the overall trend in all predictors and according to experimental data is the same. According to literature on experimental analyses of the de novo proteins [Lange et al., 2021, Bungard et al., 2017, Matsuo et al., 2021, Her et al., 2019], the de novo proteins can be ordered by estimated amount of disorder to verify comparability of the different predictors. Bsc4 contains the least disordered residues, followed by NCYM with around half of the residues in disordered regions. Goddard is highly disordered, containing only one (long) helix, while AFGP has the highest amount of disorder among the here discussed de novo proteins. In most computational de novo protein studies, either the mean or the fraction are reported to use as a comparison between different classes of de novo proteins [Xie et al., 2019, Schmitz et al., 2018, Dowling et al., 2020, Wilson et al., 2017]. When comparing these single disorder values for the de novo proteins at hand, only results from the fraction of residues in a disordered region predicted by flDPnn correspond to the experimental data. Overall, flDPnn slightly outperforms IUPred when comparing the disorder predictions with the experimentally characterised structures of de novo proteins. The same was observed in CAID [Necci et al., 2021] where disorder predictions are assessed based on recently determined structures containing disordered regions. Equally, flDPnn predicted the right order from low to high disorder in the control proteins while IUPredS and IUPredL did not (see extended materials Figure 2). However, all three predictors resulted in lower disorder values for the highly disordered proteins than indicated by experimental data. The control proteins Nop10, AF9 and synuclein are mostly disordered proteins with over 67 % to 100 % of residues in disordered regions. All three predictors results in lower percentage of disordered regions predicted ranging from 28 % (IUPredL for Nop10) to 68 % (flDPnn for synuclein). The predictions of the control proteins with both homologous sequences as well as experimentally determined structures available, are close to the experimentally observed disorder for the more structured proteins. For cytochrome and the hydrolase all three predictors resulted in percentages of disorder close to zero in accordance with experimentally determined structures (see Figure 2). Predictions of the control proteins with high disorder were lower than observed experimentally, but nevertheless the order of proteins from low to high disorder between observed and flDPnn was the same. This indicates that not only the orphan status of de novo proteins pose a problem for disorder predictors. Also, the high amount of disorder that is a commonly associated trait in de novo proteins may be one of the hurdles in disorder prediction of de novo and orphan proteins. Therefore, for the prediction of protein disorder in orphan proteins, such as de novo proteins, or other proteins without homologous sequences available, like random sequence proteins or designed proteins, still more suitable predictors are needed. In the absence of such more applicable predictors, it seems advisable to obtain and provide, wherever possible, additional experimental evidence on structure.\n\nPredicted disorder for de novo proteins by IUPred, the most widely used program, differs significantly i) between results when used âshortâ vs. âlongâ prediction parameter and ii) to results from flDPnn, which is among the best disorder predictors according to CAID [Necci et al., 2021, Hu et al., 2021]). While most studies on de novo proteins use IUPred, the use of âlongâ and âshortâ prediction varies from study to study, as well as the type of value (mean/median probability or fraction of disordered residues) that is eventually reported for comparison [Schmitz et al., 2018, Wilson et al., 2017]. This poses another problem of comparability between different studies on de novo proteins. While most studies on de novo proteins use IUPred, there seems to be a disagreement whether the âlongâ or âshortâ parameter is most suitable. According to the authors of IUPred [Dosztányi et al., 2005, ErdÅs et al., 2021], âshort\" disorder is used for small patches of disorder, for example in partially solved X-ray structures and generally predicts higher disorder at the N- and C-termini. Therefore, the same residues are predicted differently when placed at the termini of a sequence, rather than towards the centre. The âlongâ option should be used for global disorder in a protein. Accordingly, the âlongâ parameter prediction seems best suitable for predicting disorder if IUPred is deployed to de novo proteins. However, most studies favour the âshortâ prediction [Lange et al., 2021, Bungard et al., 2017, Schmitz et al., 2018, Dowling et al., 2020] over the âlongâ prediction [Xie et al., 2019]. Only few studies use both [Basile et al., 2017], while others do not state explicitly which one was used [Baalsrud et al., 2018, Wilson et al., 2017]. In these cases it must be assumed that the default âlongâ was applied.\n\nLike other disorder and predictors, IUPredâs output assigns a probability for an amino acid being in a disordered region. A protein sequence of 100 amino acids accordingly results in 100 single probabilities for disorder. For easier comparison between multiple proteins, most studies [Schmitz et al., 2018, Wilson et al., 2017, Xie et al., 2019] only report a single value per protein sequence, instead of the probabilities per residue. This reported value can either be the fraction of residues predicted to be in a disordered region [Schmitz et al., 2018, Dowling et al., 2020, Basile et al., 2017], or the average or median value of probability for disorder over the whole sequence [Wilson et al., 2017, Xie et al., 2019, Baalsrud et al., 2018]. The reported values of predicted disorder per protein reported within a study usually do not affect the analyses [Schmitz et al., 2018]. However, the combination between different parameters chosen and different values reported for the disorder per protein makes it difficult to accurately compare results of disorder predictions between studies.\n\nTherefore, both values (fraction and mean) should be reported when comparing disorder values between proteins as done for example in Eicholt et al. (2022). Similarly, when analysing single proteins, it is recommended to use multiple disorder predictors as implied for example in the MPI toolkit [Alva et al., 2016]. For bulk comparisons between different sets of proteins this is not always possible. Therefore, the disorder algorithm should be chosen carefully with provision of insights from the most recent structure prediction assessment [Necci et al., 2021]. However, all compared predictors here failed to predict disorder accurately compared to experimental evidence. The predictions of the control proteins with both homologous sequences as well as experimentally determined structures available, are close to the experimentally observed disorder for the more structured proteins. Predictions of the control proteins with high disorder were lower than observed experimentally, but nevertheless the order of proteins from low to high disorder between observed and flDPnn was the same. This indicates that not only the orphan status of de novo proteins pose a problem for disorder predictors. Also, the high amount of disorder that is a commonly associated trait in de novo proteins may be one of the hurdles in disorder prediction of de novo and orphan proteins. Therefore, for the prediction of protein disorder in orphan proteins, such as de novo proteins, or other proteins without homologous sequences available, like random sequence proteins or designed proteins, still more suitable predictors are needed. In the absence of such more applicable predictors, it seems advisable to obtain and provide, wherever possible, additional experimental evidence on structure.\n\nAF2 relies on an MSA to detect co-evolutionary patterns which is utilised to indicate the proximity of residues in space. More recently, protein language models, so called pLMs, have been employed for protein structure predictions. Structure predictors based on pLMs, such as Omegafold (OF) [Wu et al., 2022a], ESMfold [Lin et al., 2022a] and RGN2 [Chowdhury et al., 2022a] are trained to fill in the blanks of masked sequences, thereby learning interconnections between residues in protein sequences [Michaud et al., 2022]. This training is analogous to gap-filling exercises when learning a new language [Ferruz and Höcker, 2022, Ofer et al., 2021]. OF, ESMfold and RGN2 combine their language models with the structure module of AF2. In their original publications, the three pLM predictors were also tested on a set of orphans and compared to the performance of AF2 on the same set. Nevertheless, in all studies a different depth of search was employed to classify sequences as orphans. For OF, recent additions to the PDB without homologs were selected. For ESMfold also recent additions to the PDB were selected, clustered with mmseqs (70% identity threshold) and HHblits was used to confirm zero hits. Additionally, AF2âs MSA generation on UniRef, MGnify and BFD was used to find sequences with <1,<10,<100 hits, and TMscore<0.5 for any structural template. TM score is a metric used to assess the similarity of two protein structures encoded by the same sequence, while TMscore=1 indicates identical structures [Zhang and Skolnick, 2004]. For RGN2, orphans were defined as sequences with MSAdepth=1 across UniRef30, PDB70 and MGnify, meaning the resulting MSA consists only of the query sequence but no other [Chowdhury et al., 2022a]. Only OF and RGN2 were tested on orphans with experimentally determined structures and predictions compared to those of AF2. While OF outperformed AF2 significantly in that OF predictions had higher TM-scores when compared to experimentally solved structures, RGN2 surpassed AF2 only slightly, which might be due to several reasons. First, OFâs ability to predict orphans was tested on recently solved structures that were neither part of the OF training set nor of the AF2 training set, while in the case for RGN2 the majority of orphans were in the training for both [Ahdritz et al., 2022, Chowdhury et al., 2022a]. Second, the definition of orphans as consisting of an MSAdepth=1 in RGN2 might cause a bias towards short proteins and AF2 might be able to solve the global search problem for the energy minima of these short proteins despite AF2 not being optimised for short de novo proteins [Eicholt et al., 2022]. While RGN2 and OF are based on different pLMs (AminoBERT [Chowdhury et al., 2022a] and OmegaPLM [Wu et al., 2022a]) both programs employ a geometry based module before feeding into the AF2 structure module. Due to this similar architecture of OF and RGN2, we would expect the performance of OF and RGN2 on structure prediction of orphans to improve in a similar manner in comparison to AF2. We assume that the overlap of training and chosen test set in the RGN2 study might have led to an overestimation of the accuracy for both RGN2 and AF2 on the respective set of orphans. According to its original study [Lin et al., 2022a], ESMfold performed less accurately than AF2 on orphans and on proteins with an MSAdepth<10 and <100. Nonetheless, one major advantage of pLM based predictors is speed. AF2 already decreased the runtime of predictions from formerly weeks on ab initio structure prediction servers (such as QUARK or Rosetta [Xu and Zhang, 2012, Rohl et al., 2004]) to minutes. Yet, pLM based approaches promise to be multiple times faster than AF2 by skipping the computationally expensive MSA generation. Nevertheless, a language model would have to be retrained on high computing resources to stay up to date with continuously growing sequence and structure databases.\n\nFor AFGP the presence of α-helices and high disorder is also approximated by experimental studies but none of the programs predicts the polyproline II-helices suggested by experiments [Her et al., 2019]. One obstacle for structure prediction of highly dynamic proteins that becomes apparent here is the lack of prediction of ensembles of conformations. Absence of conformational ensembles is a general problem of predictions, experimental determination and deposits in the PDB [Alderson et al., 2022, Saldaño et al., 2022, Lindorff-Larsen and Kragelund, 2021]. This could, for example, lead to a wrong estimation of disorder levels [Ruff and Pappu, 2021]. Interestingly, for AFGP in D. mawsoni, AF2 is able to predict the experimentally confirmed structures [Giubertoni et al., 2019]. AF2 predicts a polyproline II-helix for the peptide [Giubertoni et al., 2019] and for the polypeptide the β-solenoid structure composed of T-(A/P)-A tandem repeats (see underlying data). This AFGP from D. mawsoni has not emerged de novo but from a serine protease, while being composed of the same repetitive structure as de novo emerged AFGP.\n\nIn the case of Bsc4, only the two pLM based programms (OF and ESMfold) are predicting the larger β-sheets determined by experiments [Bungard et al., 2017] and both predict an α-helix around the same position as AF2 and RGN2 (K62-R83). In this case, a prediction with lower pLDDT might actually be closer to reality or reflect the conformational heterogenity arising from structural dynamics [Del Alamo et al., 2022, Saldaño et al., 2022].\n\nWhile it may be reassuring that all approaches predict the structure of Goddard effectively the same, the difference in pLDDT shows how pLDDT values may differ between programs while the underlying structure predictions do not.\n\nFor NCYM, the α-helix predicted by OF and AF2 are longer than determined by experiments (A46-G59) [Matsuo et al., 2021]. The one α-helix predicted by RGN2 is shorter but in the correct position. The second α-helix predicted by OF is not supported by experimental data [Matsuo et al., 2021]. RGN2 is the only method that predicts β-sheets which are also in the positions supported by experiments (R60-C64, C104-I107). Other β-sheets suggested by experiments were not predicted by any of the programs. This indicates that, when comparing the results of different structure prediction programs, the prediction with the highest confidence score is not necessarily the most suitable one.\n\nDifference in pLDDT scoring while predicting both similar or different structures can become problematic. Especially, when pLLDT is used as a proxy metric in bulk studies [Bruley et al., 2022, Wilson et al., 2022, Tunyasuvunakool et al., 2021, Akdel et al., 2022]. A switch in structure predictor would possibly lead to very different pLDDT values. Different programs could potentially predict the same structures while the pLDDT output is different as in the case for Goddard. As for AFGP, different pLDDT with the same predicted disorder can be obtained from different predictors. For Bsc4, only OF and ESMfold were capable of predicting β-sheets that were deduced experimentally, while for NCYM only RGN2 predicted correctly the experimentally confirmed β-sheets and the α-helix in the correct position. Only for Goddard all predicted structures were in agreement. While such an agreement did not apply to all pLM based approaches, all three were capable of predicting confirmed secondary elements that AF2 could not. In general, the selected de novo proteins with their structural heterogeneity, isolation in sequence space and disorder levels are a challenge for any prediction program. All predictions have an average low (70> pLDDT >50) to very low (<50 pLDDT) confidence. Such a low confidence can be an indicator of disorder and/or of low-quality MSAs [Bordin et al., 2022]. Both disorder and low-quality MSAs are respectively a proposed property and a hallmark of de novo emerged proteins [Bornberg-Bauer et al., 2021].\n\nWhen comparing the structure predictions of de novo proteins to the ones of evolutionary conserved IDPs, the results indicate that the lower mean pLDDT for de novo protein predictions is not solely caused by high disorder levels (Figure 4 & Table 2). Evolutionary conserved IDPs, which exhibit high disorder levels, still have a higher average pLDDT score for each prediction program (see Table 2). This higher mean pLDDT can be attributed to higher pLDDT for the secondary elements of the evolutionary conserved IDPs than for the secondary elements of de novo proteins. The findings also highlight that the prediction of secondary elements can be consistent among different prediction programs for conserved IDPs, while pLDDT varies significantly between programs, as it is also the case for de novo protein structure predictions (Figures 4 & 3). The significantly higher pLDDT for predictions by AF2 for smaller proteins such as Goddard and Nop10 could be due to an easier global search problem for the energy minima of those smaller proteins.\n\nThe vast majority of all known proteins can be clustered into families, based on similarity of their folds, sequences and functions [Chothia, 1992]. While members of these protein families presumably share ancestry, de novo proteins represent special cases as they do not seem to fit in any evolutionary established family. Each protein family or class of folds can be seen as small islands in a vast ocean of viable sequences and proteins [Tretyachenko et al., 2022]. Only few of these islands have surfaced during the course of evolution while most remained submerged or plunged. De novo proteins can be seen as new islands, mutationally distant from all other islands in this ocean of sequences and could therefore provide unique structures and folds. Completely unique structures could further confirm de novo status of proteins for which no homologous sequences can be found in closely related genomes, since structure is more conserved than sequence [IllergÃ¥rd et al., 2009]. Also, entirely new structures are highly unlikely to derive from an ancestral protein homologous in sequence but structurally different [IllergÃ¥rd et al., 2009; Chothia and Lesk, 1986]. Novel folds were also rarely identified within new experimentally solved structures [Tóth-Petróczy and Tawfik, 2014] but recent advancements will increase dramatically the structural coverage of the known sequence space and could lead to identification and definition of new protein folds and families [Liu et al., 2022, Varadi et al., 2021, Bordin et al., 2023]. These advancements also provide new opportunities to search for structural homology of de novo proteins on a larger set of protein structures with popular structure homology algorithms already including predictions [van Kempen et al., 2022; La et al., 2009; Holm, 2022; Aderinwale et al., 2022]. While we share the general enthusiasm of these recent advancements, it remains to be decided which structure predictors will be most suitable for de novo proteins. Eventually, accuracy can only be confirmed when de novo protein structures are solved experimentally, ideally with NMR [Eicholt et al., 2022]. A key issue here is the unknown and potentially very large mutational distance of de novo proteins to the âislandsâ of protein families, i.e. the known realm within the vast sequence space. This accounts for any structure prediction approach, whether MSA or pLM based. Structures will only be predicted reliably if the sequences in training sets are close enough in sequence space to de novo proteins. Also, leveraging machine learning approaches for MSA generation could in general improve predictions for proteins with only remote partial homology to others [Llinares-López et al., 2022; Petti et al., 2022]. Vice versa, such advancements in homology search for structure predictions could be employed for improved detection and confirmation of de novo emerged proteins. Additionally, it should be kept in mind that pLDDT, when used as a proxy metric for bulk analysis, can vary drastically between the different programs (Figure 3) and is not practical to compare the actual confidence of different structure prediction programs to each other. Finally, the differences seen here between structure predictions and experimental approximations indicate that a decision for which predictors to use has to be made on a case-by-case basis for every de novo protein. Modular, open-source architectures such as OpenFold [Ahdritz et al., 2022] might allow better customization and help deciding which model is the most useful for de novo proteins. Also, multiple models using MSA and pLM could be combined to obtain larger sampling of sequences. Yet without further experimental structure determination, in the words of George E. P. Box, âall models are wrong, but some are usefulâ [Box, 1976].",
"appendix": "Data and software availability\n\nZenodo: Assesing structure and disorder predictions tools for de novo emerged proteins in the age of machine learning https://doi.org/10.5281/zenodo.7615407.\n\nThis project contains the following underling data:\n\n⢠afgp_AF2.pdb (prediction of AFGP by AF2)\n\n⢠afgp_ESM.pdb (prediction of AFGP by ESMfold)\n\n⢠afgp_OF.pdb (prediction of AFGP by OF)\n\n⢠afgp_plddt.csv (list of pLDDT for each residue of each prediction of AFGP)\n\n⢠AFGP_polyprotein_antarctic_cod.pdb (prediction of AFGP (polyprotein) from antarctic cod by AF2)\n\n⢠afgp_rgn2.pdb (prediction of AFGP by RGN2)\n\n⢠Antifreeze_glycopeptide_antarctic_cod.pdb (prediction of AFGP (peptide) from antarctic cod by AF2)\n\n⢠bsc4_AF2.pdb (prediction of Bsc4 by AF2)\n\n⢠bsc4_AF2_ranked2.pdb (prediction (2nd highest ranked of Bsc4 by AF2)\n\n⢠bsc4_ESMfold.pdb (prediction of Bsc4 by ESMfold)\n\n⢠bsc4_OF.pdb (prediction of Bsc4 by OF)\n\n⢠bsc4_plddt.csv (list of pLDDT for each residue of each prediction of Bsc4)\n\n⢠bsc4_RGN2.pdb (prediction of Bsc4 by RGN2)\n\n⢠disorder_lineplots.pdf (lineplots of disorder predictions)\n\n⢠disorder_values.csv (list of disorder values for each prediction of each protein)\n\n⢠gdrd_AF2.pdb (prediction of Goddard by AF2)\n\n⢠gdrd_ESMfold.pdb (prediction of Goddard by ESMfold)\n\n⢠gdrd_OF.pdb (prediction of Goddard by OF)\n\n⢠gdrd_plddt.csv (list of pLDDT for each residue of each prediction of Goddard)\n\n⢠gdrd_RGN2.pdb (prediction of Goddard by RGN2)\n\n⢠ncym_AF2.pdb (prediction of ncym by AF2)\n\n⢠ncym_OF.pdb (prediction of ncym by OF)\n\n⢠ncym_plddt.csv (list of pLDDT for each residue of each prediction of ncym)\n\n⢠ncym_RGN2.pdb (prediction of ncym by RGN2)\n\n⢠nycm_ESMfold.pdb (prediction of ncym by ESMfold)\n\n⢠p-values_all.csv (p-values of all statistical analyses)\n\n⢠denovo_sequences.fasta (amino acid sequences of analysed de novo proteins)\n\n⢠1Y2Y_AF2.pdb (prediction of Nop10 by AF2)\n\n⢠1Y2Y_OF.pdb (prediction of Nop10 by OF)\n\n⢠1Y2Y_plddt.csv (list of pLDDT for each residue of each prediction of Nop10)\n\n⢠1Y2Y_RGN2.pdb (prediction of Nop10 by RGN2)\n\n⢠1Y2Y_ESMfold.pdb (prediction of Nop10 by ESMfold)\n\n⢠2LM0_AF2.pdb (prediction of AF9 by AF2)\n\n⢠2LM0_OF.pdb (prediction of AF9 by OF)\n\n⢠2LM0_plddt.csv (list of pLDDT for each residue of each prediction of AF9)\n\n⢠2LM0_RGN2.pdb (prediction of AF9 by RGN2)\n\n⢠2LM0_ESMfold.pdb (prediction of AF9 by ESMfold)\n\n⢠alpha_synuclein_AF2.pdb (prediction of alphasynuclein by AF2)\n\n⢠alpha_synuclein_OF.pdb (prediction of alphasynuclein by OF)\n\n⢠alpha_synuclein_plddt.csv (list of pLDDT for each residue of each prediction of alphasynuclein)\n\n⢠alpha_synuclein_RGN2.pdb (prediction of alphasynuclein by RGN2)\n\n⢠alpha_synuclein_ESMfold.pdb (prediction of alphasynuclein by ESMfold)\n\n⢠p53_AF2.pdb (prediction of p53 by AF2)\n\n⢠p53_OF.pdb (prediction of p53 by OF)\n\n⢠p53_plddt.csv (list of pLDDT for each residue of each prediction of p53)\n\n⢠p53_RGN2.pdb (prediction of p53 by RGN2)\n\n⢠p53_ESMfold.pdb (prediction of p53 by ESMfold)\n\n⢠globular_controls.fasta (sequences of globular controls)\n\n⢠idp_controls.fasta (sequences of disordered controls)\n\n⢠mean_plddt.ods (Mean values of pLDDTs)\n\nSRQR checklist for âAssessing structure and disorder prediction tools for de novo emerged proteins in the age of machine learning are deposited on Zenodoâ: https://doi.org/10.5281/zenodo.7615407\n\n- Aubel_SRQR_checklist.pdf.\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\nAll scripts and code used are deposited on Zenodo: https://doi.org/10.5281/zenodo.7615407.\n\n⢠plddt_plotting.py (python script for plotting of pLDDT values)\n\n⢠count_aa.py (python script to count amino acids in multiline fasta file)\n\n⢠R_stats_plots.txt (Code used in R Studio to perform statistical tests and plot disorder values)\n\n⢠af2_palma_sbatch.sh\n\n\nAcknowledgements\n\nWe thank Andreas Lange for helpful comments on the manuscript and Alun Jones for advice on statistical tests.\n\n\nReferences\n\nZuckerkandl E: The appearance of new structures and functions in proteins during evolution. 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Methods in enzymology. Elsevier; 2004; volume 383. : pages 66â93.\n\nSaldaño T, Escobedo N, Marchetti J, et al.: Impact of protein conformational diversity on alphafold predictions. Bioinformatics. 2022; 38(10): 2742â2748. PubMed Abstract | Publisher Full Text\n\nDel Alamo D, Sala D, Mchaourab HS, et al.: Sampling alternative conformational states of transporters and receptors with alphafold2. elife. 2022; 11: e75751. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilson CJ, Choy W-Y, Karttunen M: Alphafold2: A role for disordered protein/region prediction? Int. J. Mol. Sci. 2022; 23(9). PubMed Abstract | Publisher Full Text | Free Full Text\n\nTunyasuvunakool K, Adler J, Zachary W, et al.: Highly accurate protein structure prediction for the human proteome. Nature. 2021; 596(7873): 590â596. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBordin N, Dallago C, Heinzinger M, et al.: Novel machine learning approaches revolutionize protein knowledge. Trends Biochem. Sci. 2022; 48: 345â359. Publisher Full Text\n\nChothia C: One thousand families for the molecular biologist. Nature. 1992; 357(6379): 543â544. PubMed Abstract | Publisher Full Text\n\nTretyachenko V, VymÄtal J, Neuwirthová T, et al.: Modern and prebiotic amino acids support distinct structural profiles in proteins. Open Biol. 2022; 12: 220040. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIllergÃ¥rd K, Ardell DH, Elofsson A: Structure is three to ten times more conserved than sequence: Study of structural response in protein cores. Proteins Struct. Funct. Bioinforma. 2009; 77(3): 499â508. PubMed Abstract | Publisher Full Text\n\nChothia C, Lesk A: The relation between the divergence of sequence and structure in proteins. EMBO J. April 1986; 5(4): 823â826. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTóth-Petróczy Ã, Tawfik DS: The robustness and innovability of protein folds. Curr. Opin. Struct. Biol. 2014; 26: 131â138. PubMed Abstract | Publisher Full Text\n\nLiu J, Yuan R, Shao W, et al.: Do newly born orphan proteins resemble never born proteins? a study using deep learning algorithms. bioRxiv. 2022. preprint.\n\nBordin N, Sillitoe I, Nallapareddy V, et al.: AlphaFold2 reveals commonalities and novelties in protein structure space for 21 model organisms. Communications Biology. 2023; 6(1): 160. 2399-3642. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Kempen M , Kim S, Tumescheit C, et al.: Foldseek: fast and accurate protein structure search. bioRxiv. 2022.\n\nLa D, Esquivel-RodrÃguez J, Venkatraman V, et al.: 3d-surfer: software for high-throughput protein surface comparison and analysis. Bioinformatics. 2009; 25(21): 2843â2844. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolm L: Dali server: structural unification of protein families. Nucleic Acids Res. 2022; 50(1): 210â215.\n\nAderinwale T, Bharadwaj V, Christoffer C, et al.: Real-time structure search and structure classification for alphafold protein models. Communications biology. 2022; 5(1): 1â12. Publisher Full Text\n\nLlinares-López F, Berthet Q, Blondel M, et al.: Deep embedding and alignment of protein sequences. Nat. Methods. 2022; 1â8.\n\nPetti S, Bhattacharya N, Rao R, et al.: End-to-end learning of multiple sequence alignments with differentiable smith-waterman. bioRxiv. 2022; pages 2010â21.\n\nBox GEP: Science and statistics. J. Am. Stat. Assoc. 1976; 71(356): 791â799. Publisher Full Text"
}
|
[
{
"id": "168293",
"date": "21 Apr 2023",
"name": "Caroline Weisman",
"expertise": [
"Reviewer Expertise De novo genes",
"computational genomics. No expertise in structure prediction."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments\nThe aim of this paper is to assess the suitability of modern protein structure prediction methods for use on orphan and de novo proteins. If structure prediction methods could reliably be used for these proteins, much headway could be made in generating hypotheses as to their functions and possible origins, via computationally assessing whether their structures match those of any known, conserved proteins. As this work from these authors and others has shown, experimental characterization, both functional and structural, of this class of proteins is difficult, and so computational characterization is especially valuable.\nThere are several reasons to worry that structure prediction tools may not be well-suited for these proteins, and therefore to think that a direct test of the issue is essential. There is a general concern that, because they are relatively rare among proteins and not often the subject of primary thrusts of biological interest, they have not been much included in standard benchmarking datasets for these tools. Combined with the strong possibility that structural or other features of these proteins relevant to the structure prediction methods may be different than those of conserved proteins, this motivates a worry that methods benchmarked on conserved proteins will not generalize well to this class of proteins. There is also a much more specific and acute concern motivated by the methodology of a subset of these tools: as the authors note, tools like AlphaFold use alignments comprised of homologous sequences of the proteins, which are definitionally unavailable or highly restricted for orphans and de novo proteins. It is thus entirely unclear whether structure prediction methods will make correct predictions for orphan and de novo proteins.\nThe goal of this paper is therefore extremely well-motivated and of broad general interest for the field. Before we rush to apply these methods and take their results at face value, as is already happening, we should first pause to assess whether their results are at all accurate. I am grateful to the authors for having tackled the issue here.\nThe authors take the general approach of using the small number of these proteins for which there is some amount of convincing experimental evidence regarding their structure, and performing a careful, small-N case study to assess two things: a) how well the structures of these proteins predicted by these tools line up with the experimental reality; and b) to what extent the predicted structures from these tools agree with one another. This seems to be a well-motivated and useful way of getting at the issue.\nMost of my suggestions concern the organization of the manuscript, as I think that the results are actually more important than the current structure allows one to easily see at present. I found the paper difficult to follow due to its organization and to the particular results that were presented (and if I have misunderstood the aims themselves, clarification in the text would be useful). I also wonder about including some additional comparisons or analyses that strike me as relevant to these central questions and that could be easily produced from the data that already exist in the manuscript.\nSuggestions for reorganization\nI found the flow of results in the manuscript to be a little confusing and scattered. It is not clear to me what the overall organizational paradigm in their presentation is; I felt somehow pulled back and forth. Clarification here would help.\nA suggestion for a structure that would have worked better for me is to divide the manuscript into two parts, corresponding to the a) and b) aims that I described above. So, for example, Figure 1, which compares the results of the disorder prediction methods to one another, would fall into a section corresponding to a); and Figure 2, which compares predictions to experimental ground truths, would fall into b); Figures 3 and 4, which compare methods to one another, into a).\nHighlighting central analyses by moving from the Discussion to Results and adding figures\nThis organization makes salient what I feel to be two missing analyses that could be produced from the results already in the manuscript.\nFirst, to me, the most important analysis is the comparison of the existing experimental structure data for the de novo/orphan proteins to the structures predicted by these programs. As the authors note, these existing data are not fully-solved structures, so that a traditional e.g., RMSD/visual overlay analysis cannot be performed. Nonetheless, qualitative, heuristic comparisons can and should be done. Indeed, the authors do this in the Discussion section. But I think this should be moved to Results under its own heading: it is arguably the key result of the paper. Perhaps the authors are trying to be conservative in calling it a Result, as it is qualitative and therefore perhaps feels subjective â but I disagree! One could systematize this analysis to some extent by e.g., making a table to systematically carry out the qualitative comparisons that they describe in the text. This kind of analysis is what I expect upon reading the title and abstract of this paper, and so I feel strongly that to bury it in the Discussion section with little fanfare does not do it justice. Similarly, some kind of overall summary â the punch line of, in their estimation, how well these structures have been predicted â would be useful. (A change in the title to this effect to describe the actual results of the assessment could constitute this.)\nSecond, I am not sure why Figure 2 does not include results for predicted and experimentally characterized disorder content for the de novo/orphan genes. This is another result that is described verbally in the text of the Discussion, but for the same reasons as above strikes me as a key result, and could easily be included on the axes of Figure 2.\nAdding a summary/bottom line The authors may again be trying to be conservative and not overstate conclusions from their analyses, as is their prerogative. But I would like to note that, as a reader, I do not feel that an overall summary or punch line would have been misplaced. There are hints of this, but they are again buried in the Discussion; for example, the sentence âWhen comparing these single disorder values for the de novo proteins at hand, only results from the fraction of residues in a disordered region predicted by flDPnn correspond to the experimental dataâ strikes me as an enormously important conclusion, but is nowhere in the Results, is not emphasized beyond this one-off comment, and so could easily be missed by the casual reader. I think this kind of telegraphic conclusion is important for the results being recognized and taken up by the field.\nThe authors have done much work to benchmark these methods. Some kind of summary statement in answer to the actual question motivating the work -- how well do these methods perform on de novo protein structures? -- feels apt and missing. The title of the MS would be one place to send this message. A dedicated section in the Results containing conclusions like the one above would be another.\n\nSimilarly, the finding about parameter sensitivity for disorder is extremely important and addresses a major issue in the field: this parameter is often different between studies and is sometimes not even reported. This is an important call to attention for the field. The analysis of which metrics for agreement or disagreement are reported and how they lead to very different conclusions is extremely important for the same reason. This result is more clearly articulated in the Results, but also bears repeating in a section like the one proposed.\nAdditional questions/comments\nFollowing are some more minor questions about the scientific content and analyses in the text.\nThe MS says, and this strikes me as correct, that pLDDT should not be compared between prediction methods â but much of the paper seems to do just that (the final paragraph before discussion and the whole right panel of Figures 3 and 4). I am confused as to the apparent contradiction. Similarly, is it meaningful to say that there are fewer significant differences in pLDDT for conserved IDPs than de novo genes? Is the meaningful comparison not something about the similarity or differences between the predicted structures themselves (left panel of Figures 3 and 4)?\n\nWhy does the order of disorderedness in a set of proteins (which protein has most vs least) matter? Iâm not sure this is a useful metric to be reporting. There is a reference to previous literature, but it does not explain why this is an informative metric.\n\nIf the suggested plot of predicted vs observed disorder for de novo/orphan proteins (like Figure 2) were included, there may be a clear answer to a question that emerges: the data suggest pretty strongly that disorder prediction programs underestimate disorder in highly-disordered conserved proteins, and that they underestimate disorder in de novo proteins. Is the underestimation in de novo proteins merely due to their high disorder, or is their lack of conservation also contributing? If the quantitative degree of difference is similar to the difference for conserved proteins, the former seems likely; if it is worse, the latter seems likely. This would be useful to know.\n\nOn parameter sensitivity: insofar as IUpred has only one parameter, it seems possible to dig in a little more meaningfully. Can you say something (not necessarily an experiment; just a comment) about the use of short vs long in IUpred? What does that parameter mean? What are they intended to represent? When is one more appropriate than the other per the manual? Do you find that one agrees more with the experimental data?\n\nOn disordered residues: the comparison between average disorder and percent of disordered residues is important and well-taken, but in terms of predicting the structure, is it useful to also consider the physical distribution of disordered residues (which residues are disordered, and whether methods agree)? This seems like an important metric to me in assessing the agreement between methods. For example, in an extreme case, two methods could each predict that 50% of residues in a protein are disordered, but have 100% disagreement about which residues are disordered if they predict nonoverlapping sets of residues to be disordered. In these types of scenarios, the percentage alone may be a misleading indicator of agreement.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9683",
"date": "19 May 2023",
"name": "Margaux Aubel",
"role": "Author Response",
"response": "We thank the reviewer for the approval and useful comments. Here we respond to the comments by adding our response in bold: \"Highlighting central analyses by moving from the Discussion to Results and adding figures\"/\"Adding a summary/bottom line\" We understand the concern about organisation of the manuscript, however we followed the journal guidelines and editorial comments. Further, we keep the division between results and discussion clean as to not cause confusion about our own predictions and data previously generated by others. \"I am not sure why Figure 2 does not include results for predicted and experimentally characterized disorder content for the de novo/orphan genes. \" Unfortunately, there are no absolute numbers from the experimental data to serve as ground truth. All experimentally characterised structures of de novo proteins include predictions which are difficult to disentangle from experimental data. \"The MS says, and this strikes me as correct, that pLDDT should not be compared\" Indeed, pLDDT should not be compared on the basis that it cannot be used as an deduction which predictor is the most accurate. Nevertheless, we compare it, in connection with the predicted structures, to show how pLDDT can differ or remain similar while the predicted structure remains the same or is differently predicted. This is to raise concern especially when predictors are switched for large scale studies of predictions or to select for structures with e. g. mean pLDDT >70. \"Why does the order of disorderedness in a set of proteins (which protein has most vs least) matter?\" Multiple studies cited use this as a relative comparison between different de novo proteins and protein groups. \" Can you say something (not necessarily an experiment; just a comment) about the use of short vs long in IUpred? \" This is included in the discussion about which parameter would be best for disorder prediction of de novo proteins. \" ...is it useful to also consider the physical distribution of disordered residues (which residues are disordered, and whether methods agree)?\" We have included the corresponding line plots with per residue values in underlying data and saw that the methods agree there, which is why we did not further investigate this."
}
]
},
{
"id": "168292",
"date": "24 Apr 2023",
"name": "Isabelle Callebaut",
"expertise": [
"Reviewer Expertise Structural bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is devoted to the evaluation of disorder and structure prediction methods for the specific case of de novo proteins. Structure prediction for such proteins is a challenge since, by definition, they have no homologs, on which AI-based prediction methods such as AlphaFold2 are based. Therefore, the authors examined recently developed natural language models of proteins, used for alignment-free structure predictions, to assess their value compared to those based on taking homologs into account. They performed a comprehensive comparison of the methods, based on the consideration of four de novo proteins whose structures were studied experimentally and carefully evaluated the influence of the chosen parameters.\nThe result is a critical, thorough and well-argued analysis and discussion of the information provided by these methods and their limitations, which on the one hand provides a solid basis for anyone wishing to apply them to such cases and on the other hand highlights the complexity of the issue and the need for further development.\nMy only small suggestion would be the following: the authors mainly discuss global features through the calculation of the mean probability for disorder, the fraction of disordered residues and the mean of the pLDDT values, even though the values per amino acid are displayed at the level of each figure (with more or less dispersion reflecting the heterogeneity of the proteins in term of structural properties). In my opinion, it would be interesting to compare, at the level of each amino acid of the 4 proteins considered, the disorder and pLDDT values. Indeed, as it has been rightly commented in the discussion, low pLDDT values are not necessarily associated with disorder, but with an inability of structure prediction methods to predict order (an hypothesis that is all the more plausible here in the case of de novo proteins). A comparison of the predictions of disorder and pLDDTs at the amino acid level would therefore perhaps qualify and complete the analysis, by highlighting the potentiality of hidden or conditional order. This point deserves at least a little more discussion, if not exploration.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9682",
"date": "19 May 2023",
"name": "Margaux Aubel",
"role": "Author Response",
"response": "We thank the reviewer for their approval and suggestions. We second that the inverse correlation between pLDDT and disorder is an interesting point to further investigate. Nevertheless, our aim was to compare structure predictors and disorder predictors for de novo proteins not the interconnections between those different types of predictions. The indications of disorder in de novo proteins for other biophysical parameters is another ongoing project."
}
]
}
] | 1
|
https://f1000research.com/articles/12-347
|
https://f1000research.com/articles/12-343/v1
|
28 Mar 23
|
{
"type": "Research Article",
"title": "Comparison of BiP and HSP70i as markers of unfolded protein response (UPR) in segmental and nonsegmental vitiligo",
"authors": [
"Boedhy Setyanto",
"Handono Kalim",
"Sri Poeranto",
"Dhelya Widasmara",
"Handono Kalim",
"Sri Poeranto",
"Dhelya Widasmara"
],
"abstract": "Background: Unfolded protein response (UPR) is a misfolded protein that occurs because oxidative stress disrupts cellular redox potential that extends to the endoplasmic reticulum (ER). Binding immunoglobulin protein (BiP) and inducible heat shock protein (HSP70i) as ER chaperons play critical roles in melanocyte apoptosis. Our study aims to compare BiP and HSP70i as markers of UPR in patients with segmental vitiligo (SV) and non-segmental vitiligo (NSV). Methods: The subjects were composed of 64 patients diagnosed with vitiligo, of whom 33 had NSV and 31 had SV. Skin biopsy and immunofluorescence were performed. We used BiP and HSP70i as markers of UPR. Descriptive statistics were used to analyze the data. Results: UPR-BiP expression and UPR-HSP70i in the SV group was 2.66 ± 3.07 and 3.85 ± 4.92, respectively, with a p-value of 0.001 (<α = 0.05). In the NSV group UPR-BiP expression and UPR-HSP70i was 12.55 ± 11.85 and 14.79 ± 14.72 respectively, with a p-value of 0.001 (<α = 0.05). UPR expression using the HSP70i marker in both NSV and SV groups was higher than it was using the BiP marker. The relationship between UPR-HSP70i expression and UPR-BiP expression in the SV and NSV group was significant (p <0.05) and positive. Conclusions: HSP70i is superior to BiP as a marker for expressing UPR.",
"keywords": [
"HSP701",
"BiP",
"segmental vitiligo",
"non-segmental vitiligo"
],
"content": "Introduction\n\nSkin depigmentation that occurs over time as a result of cutaneous melanocytes is a characteristic of vitiligo, where the classification of vitiligo is divided into segmental vitiligo (SV) and non-segmental vitiligo (NSV), as well as mixed and unclassified vitiligo.1 Prevalence of the disease is 0.5â2% of the worldwide population.2 In Indonesia, 187 of the 2700 patients who visited the outpatient Dermatology and Venereology clinic of Kepanjen Hospital in South Malang between 2015 and 2019 had vitiligo, amounting to a prevalence of 7%. The peak occurrence of vitiligo is usually between the first and third decade of life and it has a psychological impact on the sufferer, although vitiligo is non-life threatening.3,4 The diagnosis of vitiligo can be made from clinical examination and additional laboratory tests are usually needed only for confirmation.5\n\nThe pathogenesis of SV, including neuropeptide, somatic mosaicism, and microvascular skin horning, which eventually cause melanocyte damage, is better known than that of NSV.6â8 Otherwise, NSV pathogenesis is still multifactorial and polygenic. The dominant factors in the NSV pathogenesis are oxidative stress and autoimmunity.6,9,10 However, the mechanisms involved in the onset and progression of vitiligo remain unclear. Several previous studies have linked endoplasmic reticulum (ER) stress, which has a relationship with oxidative and immune stress in the pathogenesis of vitiligo.11 Oxidative stress disrupts the cellular redox potential that extends to the ER, so that protein will fail to fold and its accumulation will activate a response called the unfolded protein response (UPR).11â13\n\nBinding immunoglobulin protein (BiP), an ER chaperone in the accumulation of unfolded protein, preferentially binds to unfolded protein peptides and releases the ER stress sensor to enable its spontaneous dimerization and activation.14,15 Inducible heat shock protein 70 (HSP70i) can also be secreted from the stressed live cells as a chaperone. Intracellular HSP70i will bind native protein in response to stress, to prevent misfolding dan cellular apoptosis. Some experimental animal models have confirmed the action and overexpression of HSP70i in progressive depigmentation.16â18 Thus, the presence of UPR is related to the extent and severity of vitiligo because it will eventually cause damage to and destruction of melanocyte cells.6,9,10,19 The progressive depigmentation of NSV and its unpredictable disease course makes it an important subject for research. Therefore, this study aims to compare BiP and HSP70i associated with melanocyte cell death in SV and NSV patients.\n\n\nMethods\n\nThis research has been declared to be ethically feasible by the Health Research Ethics Committee, Faculty of Medicine, Brawijaya University, dated July 28th, 2021, with approval number 218/EC/KEPK-S3/07/2021.\n\nThe subjects were composed of 64 patients diagnosed with vitiligo, who were split into two groups: 33 patients in the NSV group and 31 patients in the SV group. The diagnosis of vitiligo was established by anamnesis, clinical examination, and skin biopsy. Anamnesis identifies the presence of milky white patches. In terms of clinical examination, SV is acute and appears rapidly within a few weeks and stabilizes within 1â2 years.9 On the other hand, NSV is characterized by the expansion of lesions on both sides of the body, and the disease progression is chronic and lifelong. The skin biopsy identifies if there are melanocytes at the lesion area in the epidermis.14\n\nAll the recruited patients had attended the outpatient Dermatology and Venereology clinic at Kanjuruan Kepanjen General Hospital, Malang, East Java during the period of August 2021 to January 2022. All patientsâ vitiligo was established by anamnesis, clinical examination and skin biopsy. The patients had no prior keloid history and ranged in age from 18 and 60. Exclusion criteria included pregnant women and patients with diabetes mellitus, HIV, Cushing syndrome, active infection, trauma, and malignancy, as well as smokers and those with too much sun exposure. Subjects who met the criteria gave written informed consent as research subjects, and we recorded demographic history, clinical disease history, and family history of vitiligo.\n\nThe expression of the UPR marker was observed by skin biopsy and then an immunofluorescence examination to identify expression of BiP and HSP70i. In this research, all samples were taken from skin biopsy in the perilesional zone. Skin biopsy was taken the following way. Perilesional vitiligo was disinfected with 70% alcohol, and 0.25 ml lidocaine was applied as local anesthesia to the area to be biopsied. In this area, skin biopsy was performed using the punch biopsy method with a diameter of 3 mm, and the skin tissue was included in an Eppendorf. After completion, the wound was cleaned with 0.9% NaCl solution, had topical fucidic acid applied, and was closed using sterile gauze. All skin biopsies were performed at the Dermatology and Venereology outpatient clinic at Kanjuruhan Kepanjen General Hospital, and the samples were given to the clinical pathology department, Faculty of Medicine, Universitas Brawijaya Malang, for further processing.\n\nThe tissue was cut on a microtome (Leica RM2245) to a thickness of 2â3 mm then given a code according to the researcherâs gross code, with the aim that tissue samples were not exchanged between patients before the sample was inserted into a cassette. After that, it was processed with the Automatic Tissue Tex Processor (Thermo, STP 121-2977-1902) tool for 90 min, according to the standards of the Anatomical Pathology Laboratory of the Faculty of Medicine, Universitas Brawijaya Malang, until an alarm sounded completion. The process of blocking and cutting the tissue was performed, with the epidermis removed from the Tissue Tex Processor and the tissue blocked with paraffin according to the researcherâs gross code. The tissue was cut with a microtome tool with a thickness of 3â5 microns. Next came the deparaffination process in which the tissue was cut to a thickness of 3â5 microns and placed into four tubes. These were put in an oven for 3 min at a temperature of 70â80°C. The tissue was then placed into two tubes of xylol solution for 20 min each. After that, the tissue was put into four alcohol tubes, each for 3 min (hydration), and placed in running water for 15 min. The process and cutting of the skin biopsy tissue with a microtome and hematoxylin eosin (HE) staining was carried out at the Anatomical Pathology Laboratory. All slides were viewed using an Olympus IX71 fluorescence microscope under 40à magnification.\n\nAll slides that had been prepared were then stained with the direct immunofluorescence process. The staining process for BiP used the anti-BiP antibody kit (Abcam, rabbit polyclonal to GRP78 BiP, concentration ÎŒg at 1 mg/ml, catalog number Ab21685). Immunofluoresence processes were conducted in the biomedical laboratory at the Faculty of Medicine, Universitas Brawijaya. The slides were heated at 60°C for 60 min. Then the slides were immersed in the following solutions in sequence: first they were soaked in xylol solution for 2 à 10 min, then the slide was put into absolute ethanol for 2 à 10 min, then 90% ethanol for 1 à 5 min, then 80% ethanol for 1 à 5 min, then 70% ethanol for 1 à 5 min, and a last soak in the sterile Aquadest for 3 à 5 min.\n\nThe antigen retrieval process was carried out with citrate buffer. In the first step, the slide was immersed in a chamber containing citrate buffer at pH 6.0. Then the slide was placed in a water bath at 95oC for 20 mins. The slide was removed from the water bath after 20 mins, then allowed to cool to room temperature for about 20 mins. The slides were then cleaned three times for five mins with phosphate-buffered saline (PBS). The slides were then washed five times for one min with PBS Triton-X 100 0.2% (Sigma-Aldrich). The slide was incubated for 30 mins at room temperature with 3% Bovine Serum Albumin (BSA) andthe BSA solution was discarded. Subsequently, the slide with BiP antibody was incubated for an overnight period at 4°C. The slides with PBS were washed three times for 5 mins after an overnight incubation. Then the slide was incubated with 4â,6-diamidino-2-phenylindole (DAPI) staining solution (Abcam) 1:1000 for 5 mins. Next, the slides were washed with PBS for 3 x 5 mins. The slides were then closed with mounting medium and a cover glass. An immunofluorescence microscope (Olympus IX71 fluorescence microscope under 40à magnification) was used to view and document the expression of BiP. The next process was to analyze expression using ImageJ software version 1.53k (Wayne Rasband and contributors, National Institutes of Health, USA; RRID:SCR_003070), and the results were compared.\n\nThe staining process for HSP70 used anti-HSP70i antibody kit (Santa Cruz, catalog number Sc-32239, mouse monoclonal IgG1 κ HSP70 antibody, with concentration 200 ÎŒg/ml). Immunofluoresence processes were conducted in the biomedical laboratory at the Faculty of Medicine, Universitas Brawijaya. The slides were heated at 60°C for 60 min. Then the slides were immersed in the following solutions in sequence: first they were soaked in xylol solution for two à 10 min, then the slides were put into absolute ethanol for two à 10 min, then 90% ethanol for one à 5 min, then 80% ethanol for one à 5 min, then 70% ethanol for one à 5 min, and the last soak was in sterile Aquadest for three à 5 min.\n\nFurthermore, the antigen retrieval process was carried out with citrate buffer. The first step involved immersing the slides in a chamber containing citrate buffer at pH 6.0. Then the slides were placed in a water bath at 95oC for 20 mins. The slides were removed from the water bath after 20 mins, then allowed to cool to room temperature for 20 mins. The slides were then cleaned three times for five mins with PBS. They were then washed five times for one min with PBS Triton-X 100 0.2%. The slides were then incubated for 30 mins at room temperature with 3% Bovine Serum Albumin (BSA) and the BSA solution was then discarded. The slides were then incubated with the HSP antibody for an overnight period at 4°C. The slides were washed with PBS three times in five mins after the overnight incubation. Then the slide was incubated with DAPI 1:1000 for 5 mins. Next, the slides were washed with PBS for 3 x 5 mins. Then the slides were closed with mounting medium and cover glass. An immunofluorescence microscope was used to view and document the expression of BiP. The next process was to analyze expression using ImageJ software, and the results were compared.\n\nAll research data processing techniques were analyzed using the Statistical Product and Service Solution software, IBM SPSS Statistics 20 (RRID:SCR_019096). Descriptive statistics were used to examine the data and determine percentages, mean values, and standard deviation. We used the non-parametric Mann-Whitney U test since the data were not regularly distributed. To examine the variance between the two groups, the studentâs t-test was used. Statistical significance was defined as a p-value 0.05.\n\n\nResults\n\nThis study was composed of 64 vitiligo patients, of whom 33 had NSV and 31 had SV. The mean age was 22.32 ± 9.20 years in the SV group and 44.79 ± 11.24 years in the NSV group. Table 1 lists the characteristics of the subjects.15\n\nMedian UPR-HSP70i and UPR-BiP expression in the NSV group was found to be 14.79 ± 14.72 and 12.55 ± 11.85 respectively, with a p-value of 0.001 (<α = 0.05). Thus, it can be stated that there were differences in UPR expression when using the HSP70i and BiP markers in NSV patients. UPR expression using the HSP70i marker was higher compared with the BiP marker in the NSV group. For the SV group, we found the median UPR-HSP70i and UPR-BiP expression to be 3.85 ± 4.92 and 2.66 ± 3.07 respectively.15 Graphics of median UPR-BiP expression in NSV and SV, as well as fluorescence of UPR-BiP expression in NSV and SV are shown in Figure 1. Graphics of median UPR-HSP70i expression in NSV and SV, as well as fluorescence of UPR-HSP70i expression in NSV and SV are shown in Figure 2.\n\n15\n\n15\n\nMean UPR-HSP70i and UPR-BiP expression in the NSV group was found to be 14.79 ± 14.72 and 12.55 ± 11.85 respectively, with a p-value of 0.001 (<α = 0.05). Thus, it can be stated that there were differences in UPR expression when using the HSP70i and BiP markers in NSV patients. UPR expression using the HSP70i marker was higher compared with the BiP marker in the NSV group. In the SV group, median UPR-HSP70i and UPR-BiP expression were found to be 3.85 ± 4.92 and 2.66 ± 3.07, respectively, with a p-value of 0.001 (<α = 0.05). Thus, it might be concluded that there were differences in UPR expression when using the HSP70i and BiP markers in SV patients.15\n\n\nDiscussion\n\nThe ER in eukaryotic cells has an important role in synthesis, maturing protein, lipid metabolism, and intracellular calcium homeostasis.1,12 Physiologically, the ER ensures that proteins are folded, always oxidized around the folds, and filled with calcium. Subtoxic levels of ER-stress-induced UPR in the skin are required for normal cellular function, including differentiation.16 Melanocyte are more susceptible to oxidative stress in patients with vitiligo than in healthy individuals and melanocytes release reactive oxygen species (ROS) as a respond to stress.20 The over-production of ROS and it's accumulation triggers protein oxidation and fragmentation, lipid peroxidation, and DNA damage, which compromises cellular function.10,21 In addition, ROS also interfere with the work of the folding machinery of the ER, resulting in UPR.10 This situation leads to chronic and continual ER stress that induces UPR, which has a deleterious effect on cells, and the UPR in turn becomes mechanism of a cell death.15 The accumulation of UPR also disturbs transcription, translation, and mRNA translation in protein synthesis.22â24\n\nThe ER stress response or the so-called UPR has been widely recognized in autoimmune and inflammatory diseases, where the UPR contributes to the course of autoimmune disease through the formation of antigens during the degradation of misfolded proteins, secreting neo-antigens by apoptotic cells or impairing immune tolerance.4,25 The UPR can also be activated through an increase in unfolded protein that occurs as a result of the production of large amounts of protein during melanin synthesis.10 Intracellular oxidative emphasise that outcomes from outside shows vulnerability to chemicals and harsh environmental factors, besides failure of the intracellular systems to overcome stress, and induces targeted melanocytic destruction.26 Production of pro-inflammatory cytokines and signals that activate the innate immune system result from the ROS- and UPR-mediated death of keratinocytes and melanocytes. Then, pattern recognition receptors (PRR) and nucleic acid receptors through danger-associated molecular patterns (DAMP) recognize host-derived self-DNA/RNA from damaged cells.27\n\nThe three ER transmembrane sensor proteins control UPR, namely inositol-requiring enzyme 1 alpha (IRE1a), double-stranded RNA-dependent protein kinase-like ER kinase (PERK) and activating transcription factor (ATF) 6.16 All three sensors are primarily bound with BiP, which helps to maintain their inactive state, in non-stress conditions. The interplay between these three major arms of the UPR signaling pathways determines whether stressed cells survive or die.11 The protein is known as a binding immunoglobulin protein because it is found in the ER and is bound to secreted Ig heavy chains.28 The UPR pathway is activated when the calcium concentration in the ER is reduced, which causes a rapid buildup of unfolded/misfolded proteins and the promotion of dissociated BiP from three ER transmembrane sensor proteins: IRE1a, PERK, and ATF6.16 In addition, BiP/GRP78 levels are very high in various human cancers.29\n\nIn response to cellular stress and UPR activation, melanocytes secrete HSP70i, as many other cells do.27 HSP70i binds native proteins to prevent misfolding and cellular apoptosis. In experimental animal models, the role of HSP70i overexpression in gradual depigmentation has been demonstrated.24,30,31 The effector T cells are attracted to the dendritic cells by increasing signaling, which is induced by HSP70i.27 Under stress, DAMPs that convey danger signals cause the innate immune system to become more activated by way of pattern recognition receptors (PRR) that include TLRs, RIG-I similar receptors, and NLRs. By producing cytokines, chemokines, and antigen-presenting molecules, this mechanism has the potential to both negatively destroy melanocytes and spark adaptive immunity.18 Furthermore, it activates dendritic cells, and the activated DAMP and CD8+ T cells stimulate the immune response to again destroy melanocytes.27,32,33 Melanocyte-specific, cytotoxic CD8+ T lymphocytes are crucial for the melanocyteâs eventual targeted demise.34â36 It has been found that the severity of vitiligo disease is related to the intensity of the CD8+ reaction.25,37,38\n\nIn our study, HSP70i and BiP expression were found to be relatively higher in NSV than in SV, indicating a higher response to oxidative stress that can interfere with transcription, translation, and translation of mRNA in protein synthesis, which is related to the extent and severity of vitiligo in NSV. Thus, dissimilar to SV, melanocyte destruction in NSV is mediated over diverse, intricate, and interconnected mechanisms.26\n\nOur study showed that expression using HSP70i was higher than with BiP, both in SV and NSV. Stressed vitiligo melanocytes secrete HSP70i that induce dendritic cells to increase the expression of CD80 and CD86, that stimulate immune responses to melanocytes. In a previous study using a mouse model, adding HSP70i alone could worsen vitiligo, perhaps causing over activation of dendritic cells in the skin. Meanwhile, in a mouse model lacking HSP70i expression, there was no depigmentation in the experimental study, which indicates the role of HSP70i in vitiligo. These dendritic cells can be stimulated to express more coactivation markers like CD80 and CD86, which will amplify the immune response against melanocytes.4 As a result, HSP70i, which is overexpressed in lesional vitiligo epidermis compared to normal skin, might cause an autoimmune reaction in response to environmental stressors such oxidative stresses.4,35 Thus, HSP70i is preferred because it can serve as a link between the innate and adaptive immune systems.27\n\nThe existence of this UPR is seen as crucial because if it is not immediately addressed, it will disrupt homeostasis and protein, lipid, and DNA synthesis, which will then activate the apoptotic signaling pathway, resulting in melanocyte apoptosis. Furthermore, the apoptosis produces neo antigens that will stimulate the immune system.14,16 Melanocyte apoptosis is a programmed cell death mechanism involving endonuclease enzymes, characterized by shriveled melanocyte cells that have the characteristic pattern of a step ladder and eventually become fragmented and release apoptosis bodies.38 Precisely, CD8+ T lymphocytes play a variety of roles in mediating melanocyte death.39 The majority of melanocyte death is caused by immune cells breaching the self-tolerance as a result of melanocyte apoptosis, antigen exposure, and an inflammatory microenvironment.18 In our investigation, the SV group had a relatively lower expression of melanocyte cell apoptosis than the NSV group. Although both SV and NSV exhibit melanocyte damage, it is unknown whether the loss of melanocytes in SV is brought on by an autoimmune reaction or a cellular defect that is already present.18 However, our study is consistent with other studies showing an elevated anti-melanocyte antibody titer in NSV patients. This can be used as an important basis in the vitiligo pathogenesis.40\n\n\nConclusions\n\nIn conclusion, the appearance of UPR and oxidative stress affect the progression of vitiligo, especially in NSV. Both HSP70i and BiP expression can be UPR markers, although HSP70i is superior in expressing UPR, compared with BiP, because it represents a link between the adaptive and innate immune systems; thus, HSP70i is recommended for use in future studies. The existence of this UPR is crucial because it can activate the apoptotic signaling pathway resulting in melanocyte apoptosis. Additional studies are necessary to investigate if UPR can be used as a new target in the treatment of vitiligo.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patients.",
"appendix": "Data availability\n\nZenodo: Underlying data for âComparison of BiP and HSP70i as markers of unfolded protein response (UPR) in segmental and nonsegmental vitiligoâ. https://doi.org/10.5281/zenodo.7228247. 15\n\nThis project contains the following underlying data:\n\n⢠Data file 1: Data and Demographic Journal 2.xlsx [demographic data of subjects]\n\n⢠Data file 2: Journal 2-Statistical Data.docx [analysis data results]\n\n⢠Data file 3: UPR expression of HSP70i and BiP.docx [Figure of UPR expression]\n\n⢠Supplementary file 1: Ethical Approval Letter.pdf\n\n⢠Supplementary file 2: BiP-NSV.jpeg\n\n⢠Supplementary file 3: BiP-SV.png\n\n⢠Supplementary file 4: DAPI BiP in NSV.jpg\n\n⢠Supplementary file 5: DAPI BiP in SV.jpg\n\n⢠Supplementary file 6: DAPI HSP70i NSV.jpg\n\n⢠Supplementary file 7: DAPI HSP70i SV.jpg\n\n⢠Supplementary file 8: HSP70i-NSV.png\n\n⢠Supplementary file 9: HSP70i-SV.png\n\n⢠Supplementary file 10: DAPI only (BiP NSV).jpg\n\n⢠Supplementary file 11: DAPI only (BiP SV).jpg\n\n⢠Supplementary file 12: DAPI only (HSP70i NSV).jpg\n\n⢠Supplementary file 13: DAPI only (HSP70i SV).jpg\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nReferences\n\nBoniface K, Seneschal J, Picardo M, et al.: Vitiligo: focus on clinical aspects, immunopathogenesis, and therapy. 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}
|
[
{
"id": "191022",
"date": "15 Aug 2023",
"name": "Mitesh Dwivedi",
"expertise": [
"Reviewer Expertise Autoimmunity",
"Immunogenetics",
"Skin Immunity"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have investigated the UPR-BiP and UPR-HSP70i markers through their expression in the skin of vitiligo patients and established a correlation. The study seems interesting as it has been done for the first time by comparing these markers in two types of vitiligo. However, the study needs more clarification for few concerns raised here and includes few analyses as suggested to improve the study and its presentation.\nI suggest authors to improve introduction section. Authors must cite additional studies to back up their rationale for the study.\n\nHow did authors calculate the sample size? Sample size calculation must be mentioned in the methods section.\n\nIn the skin biopsy section, authors mention that perilesional skin sample was acquired from the study subjects. Why didnât author choose lesional skin for the experiments?\n\nAlthough authors have seen the expression of the UPR marker in skin biopsy and then an immunofluorescence examination to identify expression of BiP and HSP70i. But solely observing expression within skin section would not be enough to make any conclusion. I advise authors to also seek out for ROS mediated damage within the skin samples. Therefore, I recommend authors to include oxidative stress marker H2O2 levels within the skin samples.\n\nIt is difficult to understand Figure 2(a). I would advise authors to elaborate what they exactly mean by the figure in detail within legends.\n\nUPR pathway is activated when there is decline in calcium concentration in the endoplasmic reticulum which also contributes in the unfolded/misfolded proteins. Authors could have also look for calcium levels within the patients. Consequently, authors can cite recent studies done on calcium and calcium related effects in vitiligo1-4.\n\nWhy didnât authors see expression levels of endoplasmic reticulum sensor proteins such as ATF, IRE, PERK, etc.?\n\nHSP-70i and BiP could also play a role in the immunological mechanism leading to vitiligo through activation of lymphocytes and macrophages, activation and maturation of dendritic cells as an antigen presenting cells and initiating the release of cytokines. It would be interesting to evaluate the production of TNF-α, IL-1β, IL-12, and IL-6 within skin samples because they are apoptosis mediators and inhibitor of melanogenesis leading to vitiligo.\n\nIn addition, authors could also look for inflammatory cytokines such as IFN-gamma, IL-17 in order to relate its relation with ROS production.\n\nMoreover, authors must carry out correlation analysis for expression of HSP-70i and BiP with the disease activity and vitiligo patients.\n\nAbstract: Please remove (<α = 0.05) from results section.\n\nPlease check the sentence formation and grammatical errors throughout the manuscript: \"UPR expression using the HSP70i marker in both NSV and SV groups was higher than it was using the BiP marker.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "198869",
"date": "20 Sep 2023",
"name": "Shintaro Inoue",
"expertise": [
"Reviewer Expertise Cell biology and biochemistry (vitiligo pathophysiology",
"tight junction barrier regulation",
"and regulation of hyaluronan metabolism in the skin)"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors compared the usefulness of two markers (BiP and HSP70i) in SV and NSV and concluded that HSP70i is superior to BiP for assessing UPR. However, there is insufficient evidence to draw the authors' conclusions for the following reasons:\nThe Bip (rabbit polyclonal) and HSP70i (mouse monoclonal) antibodies have different reactivity and chromogenicity toward their respective proteins. Even in the same skin sample, you cannot compare the expression levels of Bip and HSP70i proteins by their chromogenic intensity. You can only compare the fluorescence intensity between lesional and non-lesional areas of the same patient using the same antibody, or between SV and NSV stained simultaneously using the same antibody.\n\nTo consider the significance of Bip and HSP70i in vitiligo pathogenesis, it is desirable to show differences in signal intensity between lesional and non-lesional areas in SV and NSV, respectively.\n\nI am unable to see the quantitative data that should be shown in Fig. 1a and Fig. 2a.\n\n97% of NSV and 55% of SV patients receive topical steroid treatment. At the very least, I would like to see verification that there is no difference in staining intensity of Bip and HSP70i when SV patients are divided into groups with and without topical steroids. If possible, it would be nice to have data on patients with NSV who have not received topical steroid therapy in the months prior to the study.\n\nThere is no information on the stage of disease (stable, progressive, etc.) or the analysis sites (lesion, perilesional, non-lesion) used in the analysis, nor is there any information on the sites of selection or selection criteria for the analyses.\n\nIn NSV, extracellular secretion of HSP70i is important for dendritic cell activation. It would be interesting to have a comparison of fluorescence intensity of extracellular HSP70i limited to just below the basement membrane.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-343
|
https://f1000research.com/articles/11-61/v2
|
08 Mar 22
|
{
"type": "Research Article",
"title": "The improvement of inflammatory markers and disease progression among moderate, severe, and critical COVID-19 patients: a cross-sectional study from two second referral hospitals in Surabaya, Indonesia",
"authors": [
"Pradana Zaky Romadhon",
"Siprianus Ugroseno Yudho Bintoro",
"Satriyo Dwi Suryantoro",
"Tri Pudy Asmarawati",
"Alfian Nur Rosyid",
"Merlyna Savitri",
"Putu Niken Ayu Amrita",
"Muhammad Noor Diansyah",
"Ami Ashariati Prayoga",
"Choirina Windradi",
"Bagus Aulia Mahdi",
"Krisnina Nurul Widiyastuti",
"Dwiki Novendrianto",
"Esthiningrum Dewi Agustin",
"Firas Farisi Alkaff",
"Kartika Prahasanti",
"Didi Darmahadi Dewanto",
"Pradana Zaky Romadhon",
"Tri Pudy Asmarawati",
"Alfian Nur Rosyid",
"Merlyna Savitri",
"Putu Niken Ayu Amrita",
"Muhammad Noor Diansyah",
"Ami Ashariati Prayoga",
"Choirina Windradi",
"Bagus Aulia Mahdi",
"Krisnina Nurul Widiyastuti",
"Dwiki Novendrianto",
"Esthiningrum Dewi Agustin",
"Firas Farisi Alkaff",
"Kartika Prahasanti",
"Didi Darmahadi Dewanto"
],
"abstract": "Background: To date, coronavirus diseases 2019 (COVID-19) has no definitive treatment. Thrombosis and hypercoagulation may occur in the advanced stage. Further study on how to use anticoagulants is still required to promote the best prognosis. Methods: A cross-sectional study of 110 moderate, 140 severe, and 81 critical patients receiving unfractioned heparin (UFH), low-molecular-weight heparin (LMWH), and fondaparinux was conducted. Data were collected from March 15th to August 31st 2020 at Universitas Airlangga and Husada Utama Hospital. A comparative study of white blood cell (WBC), neutrophils, lymphocytes, neutrophil-lymphocyte ratio (NLR), c-reactive protein (CRP), procalcitonin (PCT), D-dimer, all-cause mortality rate, length of stay, and days of death among three severities of COVID-19 was done. Univariate and multivariate analysis were used to determine the correlation between inflammatory state after anti-coagulant with patientsâ mortality. Results: Two deaths occurred in moderate cases, 36 deaths in severe cases, and 70 deaths in critical cases on ventilators. On day 13, moderate and severe groups showed decreased WBC, neutrophils, NLR, CRP, and D-dimer (p < 0.05). NLR, CRP, and D-dimer (p<0.05) in critically ill and ventilated patients decreased. Day-13 evaluation revealed 32.73% decrease of inflammatory markers in moderate group; 32.86% in severe patients; and 16.05% in critically ill, ventilated patients. A significant correlation between day 13 inflammatory status with mortality was seen in moderate and critical cases with a ventilator (r=0.337; p< 0.05 and r=0.25; p 0.05). Inflammatory profile on day 6 (adjusted odds ratio [aOR] = 2.36; p < 0.05) and day 13 ([aOR] = 4.15; p < 0.05) was associated with patientsâ mortality. Conclusions: Anticoagulants in COVID-19 patients lower inflammation markers. Evaluating inflammatory status is essential to predict the mortality. Inflammatory markers on day 13, based on the severity of COVID-19 and comorbidities, were associated with mortality in moderate and critical cases.",
"keywords": [
"COVID-19",
"Anti-coagulant",
"mortality",
"length of stay",
"severity",
"health"
],
"content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) infection has caused widespread novel COVID-19 pneumonia, causing respiratory problems.1 The World Health Organization (WHO) declared this as a global pandemic affecting many aspects of life.2 A severe degree of COVID-19 pneumonia is defined as a condition in patients complaining of difficulty in breathing, plus one of: respiratory rate >30 times per minute; severe respiratory distress; or oxygen saturation (SpO2) <93% in room air or a PaO2/FiO2 ratio (PF Ratio) <300. In children, it is defined as having a cough or difficulty breathing, plus at least one of: central cyanosis or SpO2 <90%; severe respiratory distress (such as snoring, heavy chest wall traction); signs of severe pneumonia, namely inability to suckle/drink; lethargy or decreased consciousness, or seizures. Other signs of pneumonia are chest retractions, rapid breathing >60Ã/minute in children aged <2 months, >50Ã/minute in children aged 2-11 months, >40Ã/minute in children aged 1-5 years, or >30Ã/minute in children aged >5 years.2\n\nSevere SARS-CoV2 infection promotes a syndrome related to prothrombotic conditions, in which blood clots easily. This condition is characterized by several specific abnormal laboratory values, such as mild thrombocytopenia, increased fibrin, degradation of fibrin products, fibrinogen, and D-dimers. Increased D-dimers are strongly correlated with worsened clinical conditions and increased risk of death in COVID-19.3,5 Based on reports from several studies, there is a growing incidence of several thromboembolic states in patients with COVID-19 admitted to the intensive care unit (ICU), one of which is pulmonary embolism. COVID-19 patients suspected of experiencing a thromboembolic event should be given anticoagulant therapy when radiological imaging is difficult to perform.3â5\n\nThe pathophysiology of hypercoagulation in COVID-19 patients is still currently being explored. A case series presenting three cases of COVID-19 with antiphospholipid syndrome has recently been reported by The New England Journal of Medicine.5 SARS-CoV-2 infection is associated with antiphospholipid antibodies, which predispose to hypercoagulation. The study of Campbell et al. reported severe COVID-19 patients with increased levels of lactate dehyrdrogenase (LDH), D-Dimer, bilirubin, decreased platelets, mild anemia, heart and kidney injury, and diffused thrombotic micro-angiopathy.5â7\n\nPatients with severe COVID-19 experience complications of coagulopathy in the form of disseminated intravascular coagulation (DIC) which might result in death. Severe COVID-19 patients experience respiratory problems and increased virulence, according to the criteria of the Third International Consensus Definitions for Sepsis (Sepsis-3). Severe COVID-19 patients are also at risk of venous thromboembolism (VTE) due to prolonged bed rest. The International Society of Thrombosis and Haemostasis (ISTH) provides a new category to identify the early phase of sepsis-associated DIC which is also called sepsis-induced coagulopathy (SIC). COVID-19 patients who fit the diagnostic criteria for SIC can be also given anticoagulant therapy.5â10\n\nAnticoagulant therapy in COVID-19 patients is administered to those who show signs of thrombosis, such as elevated inflammatory factors and D-dimers within 7-14 days, with threefold D-Dimer value. The option would be to use low molecular weight heparin (LMWH) at a dose of 100 IU/ kg twice a day, for 3-5 days. The European Society of Cardiology also includes anticoagulants in the COVID-19 therapy algorithm. For patients admitted to the ICU, the parenteral heparin drip protocol must be strictly controlled and the time value of the active prothromblastin time is 60-85 seconds. For non-ICU patients, the subcutaneous dose of enoxaprin is started at 1mg/kg twice daily. After all the completed researches, further study on how to use anticoagulants is still being analysed to promote the best prognosis.3,11,12\n\n\nMethods\n\nApproval was issued by the research ethics board Universitas Airlangga Hospital (No: 179/KEP/2020) on 2nd October 2020. The consent to participate was not applicable since our data were obtained from medical records.\n\nWe performed a retrospective cohort study with consecutive sampling among COVID-19 adult patients admitted to two referral hospitals for COVID-19, Universitas Airlangga Hospital (UAH) and Husada Utama Hospital, Surabaya, East Java, Indonesia, from March 15th 2020 to August 31st 2020. The patients included were ones in moderate, severe, or critical condition. We assessed patients referring to WHO guidelines and Indonesian Ministry of Health guidelines, and tested for SARS-CoV-2 Polymerase Chain Reaction (PCR) through oropharyngeal and nasopharyngeal swabs. Sequential chest x-ray and laboratory inflammatory marker evaluation were performed, then we did three timeframes of assessment. The first evaluation was done at the admission, followed by a second evaluation on the sixth day, and lastly evaluated before discharge or death (mean time 13 days).\n\nIn October 2020, we collected the data from medical records of those admitted to UAH and Husada Utama hospital between March 15th, 2020 to August 31st, 2020. We had 331 patients with moderate to critically ill COVID-19. Incomplete medical records were excluded. From this selection, we organized patient records into 110 moderate cases, 140 severe cases, and 81 critically ill cases with ventilator (see Figure 1).\n\nPatients were categorized as a moderate case if they had either or both of 1) signs of pneumonia; 2) O2 sat â¥93% free air. Meanwhile, patients were classified into severe cases if there were clinical signs of pneumonia, with one of the following: 1) respiratory rate (RR) >30 times per minutes; 2) severe respiratory distress; 3) oxygen saturation < 93% free air. They were grouped into critically ill if they suffered from acute respiratory distress syndrome (ARDS), sepsis, and septic shock.2\n\nThe criteria of inflammatory markers were: 1) white blood cell (WBC) >6.16x103 cells/ÎŒL;13 2) neutrophil-lymphocyte ratio (NLR) >6.5;14 3) absolute lymphocyte count ALC < 1.0x103 cells/ÎŒL;15 4) c-reactive protein (CRP) >41.8 mg/L;16 and 5) Procalcitonin > 0,07 ng/mL.16\n\nWe examined the data using SPSS version 24.0 (Chicago, IL, USA). Descriptive analysis incorporated categorical variables reported as number (percentage) and continuous variables as mean (standard deviation). We displayed categorical variables as number (%) and continuous variables as mean (standard deviation) or median (range), depending on whether the data are normally distributed. Means of Chi square or McNemar was used to assess statistical significance for dichotomous variables, while paired t-test or Wilcoxon test were used to examine continuous variables, depending on the data distribution. We compared the mean/median difference of the first to the second evaluation and the second to the third evaluations of the laboratory results, length of stay, days of death, and mortality rate according to severity of COVID-19 using ANOVA or Kruskal Wallis. Bivariate analysis between inflammatory state and mortality was conducted using Spearman. We also determined the relationship between inflammatory state after anti-coagulant with patientsâ mortality, along with some variables, which were age, gender, disease severity, and comorbidity.\n\n\nResults\n\nOf 331 patients enrolled, 200 were male and 131 were female. Patients were grouped based on the category of severity of disease and resulted in 110 moderate patients, 140 severe patients, and 81 critically ill patients with ventilator support. The average age of patients with severe COVID-19 was 57.45 ± 13.5 years. In the anticoagulant group, the most frequent comorbid factors found were diabetes mellitus (DM) (52.86%), hypertension (HT) (46.91%), and geriatric age (47.14%) while others had history of heart disease (5.45%), stroke (3.7%), and chronic kidney disease (1.82%). We evaluated inflammatory markers as baseline study data. There were significant differences in laboratory markers between each severity group (p value <0.05). The highest white blood count was 8.53 ± 5.41 à 103/L, neutrophils 83.75 ± 9.51%, lymphocyte 17.1 ± 9.9%, lymphocyte absolute 1.1 ± 0.57 à 103/L, neutrophil lymphocyte ratio (NLR) 8.38 ± 12.5, the C-reactive protein (CRP) 40.1 ± 42.43 mg/L and d-dimer 1.2 ± 8.53 mcg/L. The outcome analysis showed significant differences in the mean length of stay (p value <0.05) (see Table 1).\n\n* Kruskal Wallis.\n\nWe evaluated and compared inflammatory markers up to three times. To analyze the relationship between the administration of anticoagulants in each severity of COVID-19 cases, we calculated the decrease or increase in these inflammatory markers.\n\nBased on the first and second laboratory evaluations, there were differences in decreasing leukocyte count and D-dimer in severe cases (p 0.03; p 0.026), decreasing neutrophils neutrophil-lymphocyte ratio and CRP in moderate cases, (p 0.004; p 0.028; and p<0.05), and increasing lymphocyte in critically ill cases (p<0.05). In this initial evaluation, there were no differences in inflammatory status between each degree of COVID-19 cases.\n\nIn contrast to the initial evaluation, the comparison of the inflammatory status between the second and third examinations found that there were differences in each severity group (moderate (p<0.05); severe (p<0.05) and critically ill (p 0.001)). Inflammation marker examination found decreasing leukocytes in moderate and severe cases (p<0.05; p 0.001), decreasing neutrophils in moderate, severe and critically ill cases (p<0.05; p<0.05; p 0.007), increasing lymphocyte count in moderate cases (p<0.05); increasing absolute lymphocyte in moderate and severe cases (p<0.05; p<0.05), decreasing NLR in moderate, severe and critically ill cases (p<0.05; p<0.05; p<0.05), decreasing CRP in moderate and severe cases (p<0.05; p< 0.05), and decreasing d-dimer in all case groups (p<0.05; p<0.05; p<0.05).\n\nWith these significant results, we evaluated the inflammatory parameters from the initial to the last examination. In moderate group, there was an improvement in inflammation parameters in all variables (p<0.05). In severe cases, significant improvements in inflammatory parameters were recorded, including decreasing leukocytes, decreasing neutrophils, decreasing lymphocytes, increasing absolute lymphocytes, decreasing NLR, decreasing CRP and d-dimer (p 0.01; p<0.05; p<0.05; p<0.05; p 0.0; p<0.05; p<0.05 p<0.05).\n\nIn the critically ill with ventilator support group, significant results were also obtained on each inflammatory marker variable except for decreasing leukocytes (p 0.6). From the clinical aspect, analysis of the use of anticoagulants on day of death found significant differences among groups of severity (p<0.05). Significant correlation of inflammatory status on the thirteenth day with mortality based on patient comorbidities was seen in moderate and critical cases on a ventilator (r=0.337; p<0.05 and r=0.25; p 0.05) (see Table 2 and Figure 2).\n\n* p Value from Wilcoxon test.\n\n** p Value from Spearman correlation.\n\nUnivariate and multivariate analysis using regression logistic revealed that no decline of inflammatory profile both on day 6 (adjusted odds ratio [aOR] = 2.36; 95% CI: 1.46-3.83, p<0.05) and day 13 (adjusted odds ratio [aOR]=4.15; 95% CI: 2.33-7.42, p<0.05) was related to the mortality event (see Table 3).\n\n* p<0.05 from univariate analysis.\n\n** p<0.05 from multivariate regression logistic analysis with adjusted from potential confounder variable.\n\n\nDiscussion\n\nCOVID-19 has been linked to coagulation disorders which cause various complications. An increase in coagulation parameters, such as D-dimers, is an independent risk factor for death. Patients with D-dimers of more than 1000 ng/mL have a 20 times greater risk of death due to infection.17 Although the pathogenesis of coagulopathy in COVID-19 cannot be fully explained, the mechanism may resemble septic coagulopathy in bacterial infections. Overabundant pro-inflammatory cytokines increases the level of damage-associated molecular patterns (DAMP). Activation of coagulation factors due to cell and endothelial damage is the most common mechanism of infection. Both the pathogen and DAMP from damaged tissue activate monocytes. The activated monocytes release pro-inflammatory cytokines (IL-1, IL-6, IL-10, TNF-α) and chemokines that animate neutrophils, lymphocytes, platelets, and vascular endothelial cells. The coagulation cascade is commenced by tissue factor and phosphatidylserine on the cellsâ surface. Healthy endothelial cells retain anti-thrombogenetic properties by expressing glycocalyx and binding with anti-thrombin proteins. Damaged endothelial cells change their nature to become more procoagulant due to glycocalyx disorders and loss of anticoagulant proteins.7,12,18,19 Markers of hypercoagulation and high levels of inflammatory mediators are consistent with poor outcome in patients having acute respiratory distress syndrome (ARDS) and sepsis. These observations have led to several studies focused on inflammation and coagulation pathways in acute lung injury, ARDS or sepsis.17,20,21\n\nThe routine use of anticoagulant regimens in COVID-19 patients requiring hospitalization has been recommended, after several studies found an association between viral inflammation and coagulation disorders.22 Several guidelines for the management of COVID-19 have included anticoagulant regimens regarding COVID-19-related coagulation disorders. Recommendations issued by the Anticoagulation Forum (ACF) and the American College of Chest Physicians (ACCP) selected Low Molecular Weight Heparin (LMWH) over Unfractioned Heparin (UFH) to minimize laboratory evaluation. ACCP also recommends fondaparinux over UFH in patients with a high risk of bleeding, kidney problems, and any plans for procedures in the near future. UFH is more recommended by the ACF for patients with renal impairment with creatinine clearance <15-30 mL/min. The American Society of Hematology (ASH) states that LMWH or UFH is the therapy of choice over oral anticoagulants due to the potential for drug interactions and short half-lives.23,24 In our study, the population involved was a group of patients requiring hospitalization due to SARS-CoV2 infection ranging from moderate to critically ill. At all three severity groups, patients receiving anticoagulant therapy had increased D-dimer values. This increase in D-dimers expresses clusters of fibrin lysis and thrombus in the pulmonary vessels. Previously, Guan et al. (2020) found that 46% of 1099 COVID-19 patients had an increase in D-dimer and only 5% experienced a decrease in platelet count.19,25\n\nThe relationship between coagulation function and indicators of inflammation and infection has been previously analyzed by Long et al. (2020). D-dimers were positively correlated with CRP (r=0.36, p=0.0007) and procalcitonine (r=0.45, p<0.001).26 Increased CRP was also found in patients with coagulation disorders by Friedrich et al. (2020) to a mean level of 131 ± 106 mg/l.27 In our study, there was an increase in CRP by a mean of 40.1 ± 42.43/l in the severe group population receiving anticoagulants that met the criteria for anticoagulant according to The International Society of Thrombosis Haemostasis (ISTH) or due to an increase in D-dimer. A meta-analysis regarding dosing of anticoagulant therapy on the COVID-19 mortality rate found that there was a slight decrease in the mortality rate in COVID-19 patients in need of a ventilator.28 Several studies have shown different results on the mortality outcome of COVID-19 patients receiving anticoagulant therapy. In theory, the coagulation cascade is active when inflammation is present due to SARS-CoV2 infection. Therefore, it may be possible to obtain beneficial effects from the use of anticoagulants as anti-inflammatory agents.\n\nAccording to the theory of hypercoagulation disorders, there is a two-way relationship between the immune system and thrombin formation, where inhibition of thrombin formation may reduce the inflammatory response.29 The positive effects of using anticoagulants on mortality were reported by Nadkarni et al.; compared to non-users, recipients of anticoagulants for both therapy and prophylaxis had reduced mortality (adjusted hazard ratio [aHR] = 0.53; 95% CI: 0.45-0.62, and aHR = 0.50; 95% CI.: 0.45-0.57, respectively), and intubation rates (aHR 0.69; 95% CI: 0.51-0.94, and aHR 0.72; 95% CI: 0.58-0.89, respectively).30 Our study revealed that no reduction in inflammatory markers was significantly correlated with the mortality.\n\nSeveral other studies have classified the use of anticoagulants according to their intended use for therapeutical and prophylaxis purpose. Klok et al. (2020) found that 15% of the population needed ICU while Helms et al. (2020) revealed 25 of their patients had pulmonary embolism despite receiving anticoagulant therapy.22 Therefore, anticoagulant prophylaxis is rational. In addition, the prophylactic use of apixaban (odds ratio [OR] 0.46, p=0.001) and enoxaparin (OR=0.49, p=0.001) exhibited a significant decrease in mortality. There was also an association between therapeutic apixaban and decreased mortality rates (OR 0.57, p=0.006). Pawlowski et al. (2020) compared the effects of enoxaparin and heparin, showing that patients receiving heparin had a higher risk of death and higher ICU admission than ones in enoxaparin group (risk ratio: 6.76; 95% C.I: [3.39, 12.7]; adjusted p-value <0.0001); (risk ratio of ICU admission: 1.51; 95% C.I.: [1.12, 2.03]; adjusted p-value 0.01). Additionally, ICU and hospital length of stay were shorter in the enoxaparin population (mean ICU duration: 0.9 days [standard deviation: 2.5], mean hospital duration: 5.4 days [standard deviation: 4.3]).31\n\nIn this study, we found favorable changes in inflammatory markers such as white blood cells, neutrophils, lymphocytes, CRP, and D-dimers after the use of anticoagulants. The analysis of the results of this study has been divided based on the subgroup of the severity of COVID-19 disease. From the clinical aspect, analysis of the use of anticoagulants on day of death found significant differences among groups of severity (p<0.05). There was also a significant correlation between inflammatory status on the thirteenth day and mortality based on patient comorbidities obtained from moderate and critical cases on a ventilator (r=0.337; p< 0.05 and r=0.25; p 0.05). Univariate and multivariate analysis exhibited that no reduction in inflammatory profile on day 6 (adjusted odds ratio [aOR]=2.36; 95% CI: 1.46-3.83, p value < 0.05) and day 13 (adjusted odds ratio [aOR]=4.15; 95% CI: 2.33-7.42, p value < 0.05) were linked to the patientsâ mortality. This study is able to explain that the values of inflammatory and hypercoagulable markers go hand in hand with the severity of the patient. Although not all inflammatory markers improve after anticoagulant treatment, inflammatory variables mostly manifested good results. This study did not distinguish one regimen of anticoagulant from another; thus, it could be a confounder. Conclusively, it is necessary to carry out further subgroup analysis of the types of anticoagulants and the comparison of therapeutical effects and prophylactic use of anticoagulant towards the mortality outcome and length of stay.\n\n\nConclusion\n\nAdministration of anticoagulants to COVID-19 patients with moderate to critical presentation promoted significant outcomes of inflammatory markers which ultimately showed a statistical difference in mortality. Most of the inflammatory markers in patients improved after anticoagulant administration. Therefore, our findings confirm that the administration of anticoagulants can be optimized since they are able to work as anti-inflammatories.\n\n\nData availability\n\n\n\n\nUnderlying data\n\nfigshare: Data of Inflammatory Parameters after Anti-coagulant among Moderate, Severe, and Critically Ill COVID-19 Patients. https://doi.org/10.6084/m9.figshare.16910905.v2\n\nThis project contains the following files:\n\n- Data_antikoagulan_all_join_3.xlsx (raw data file)\n\n- Legend (Anti-coagulant_311021).docx (data key)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthors' contributions\n\nPradana Zaky Romadhon: Conceptualization, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nSiprianus Ugroseno Yudho Bintoro: Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nSatriyo Dwi Suryantoro: Conceptualization, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nTri Pudy Asmarawati: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nAlfian Nur Rosyid: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nMerlyna Savitri: Conceptualization, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nPutu Niken Ayu Amrita: Conceptualization, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nMuhammad Noor Diansyah: Conceptualization, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nAmi Ashriati Prayoga: Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Validation, Writing â Original Draft Preparation, Writing â Review & Editing\n\nChoirina Windradi: Data Curation, Investigation, Methodology, Resources, Software, Visualization; Writing â Original Draft Preparation\n\nBagus Aulia Mahdi: Conceptualization, Data Curation Formal Analysis, Investigation, Methodology, Project Administration,Resources, Software, Validation, Writing â Original Draft Preparation\n\nKrisnina Nurul Widiyastuti: Data Curation, Investigation, Methodology, Resources, Software, Visualization; Writing â Original Draft Preparation\n\nDwiki Novendrianto:: Data Curation, Investigation, Methodology, Resources, Software, Visualization; Writing â Original Draft Preparation\n\nEsthiningrum Dewi Agustin:: Data Curation, Investigation, Methodology, Resources, Software, Visualization; Writing â Original Draft Preparation\n\nFiras Farisi Alkaff: Data Curation, Investigation, Methodology, Resources, Software, Visualization; Writing â Original Draft Preparation\n\nKartika Prahasanti: Data Curation, Investigation, Methodology, Resources, Software, Visualization; Writing â Original Draft Preparation\n\nDidi Darmahadi Dewanto: Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Writing â Original Draft Preparation",
"appendix": "Acknowledgements\n\nWe would like to thanks Prof. Dr. Nasronudin dr, Sp. PD KPTI for supporting this research.\n\n\nReferences\n\nWu Y, Huang X, Sun J, et al.: Clinical Characteristics and Immune Injury Mechanisms in 71 Patients with COVID-19. mSphere. 2020; 5. PubMed Abstract | Publisher Full Text\n\nBurhan E, Susanto AD, Nasution SA, et al.: Pedoman Tatalaksana COVID-19 5OP Edisi 3 2020. Jakarta, Indonesia: PDPI, PERKI, PAPDI, PERDATIN, IDAI; 3 ed.2020 Desember; 2020. . 149 p.\n\nTang N, Bai H, Chen X, et al.: Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. Journal of Thrombosis and Haemostasis. 2020; 18(5): 1094â1099. PubMed Abstract | Publisher Full Text\n\nCampbell CM, Kahwash R: Will Complement Inhibition Be the New Target in Treating COVID-19-Related Systemic Thrombosis?. Circulation. 2020; 141(22): 1739â1741. PubMed Abstract | Publisher Full Text\n\nKichloo A, Dettloff K, Aljadah M, et al.: COVID-19 and Hypercoagulability: A Review. Clinical and Applied Thrombosis/Hemostasis. 2020; 26: 107602962096285. PubMed Abstract | Publisher Full Text\n\nAbou-Ismail MY, Diamond A, Kapoor S, et al.: The hypercoagulable state in COVID-19: Incidence, pathophysiology, and management. Thrombosis Research. 2020; 194: 101â115. PubMed Abstract | Publisher Full Text\n\nIba T, Levy JH, Levi M, et al.: Coagulopathy in COVID-19. J Thromb Haemost. Journal of Thrombosis and Haemostasis. 2020; 18: 2103â2109. PubMed Abstract | Publisher Full Text\n\nSaleh NY, Aboelghar HM, Salem SS, et al.: The severity and atypical presentations of COVID-19 infection in pediatrics. BMC Pediatrics. 2021; 21(1): 144. PubMed Abstract | Publisher Full Text\n\nVidali S, Morosetti D, Cossu E, et al.: D-dimer as an indicator of prognosis in SARS-CoV-2 infection: a systematic review. ERJ Open Research. 2020; 6: 00260â02020. Publisher Full Text\n\nFavaloro EJ, Thachil J: Reporting of D-dimer data in COVID-19: Some confusion and potential for misinformation. Clinical Chemistry and Laboratory Medicine. 2020; 58: 1191â1199. PubMed Abstract | Publisher Full Text\n\nWadaa-Allah A, Emhamed MS, Sadeq MA, et al.: Efficacy of the current investigational drugs for the treatment of COVID-19: a scoping review. Annals of Medicine. 2021; 53(1): 318â334. PubMed Abstract | Publisher Full Text\n\nBikdeli B, Madhavan MV, Jimenez D, et al.: COVID-19 and Thrombotic or Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and Follow-Up: JACC State-of-the-Art Review. Journal of the American College of Cardiology. 2020; 75(23): 2950â2973. PubMed Abstract | Publisher Full Text\n\nFeng X, Zhu B, Jiang C, et al.: Correlation between White Blood Cell Count at Admission and Mortality in COVID-19 Patients. A Retrospective Study.2020.\n\nLi X, Liu C, Mao Z, et al.: Predictive values of neutrophil-to-lymphocyte ratio on disease severity and mortality in COVID-19 patients: a systematic review and meta-analysis. Critical Care. 2020; 24(1): 647. PubMed Abstract | Publisher Full Text\n\nWagner J, DuPont A, Larson S, et al.: Absolute lymphocyte count is a prognostic marker in Covid-19: A retrospective cohort review. International Journal of Laboratory Hematology. 2020; 42(6): 761â765. PubMed Abstract | Publisher Full Text\n\nLiu F, Li L, Xu M, et al.: Prognostic value of interleukin-6, C-reactive protein, and procalcitonin in patients with COVID-19. Journal of Clinical Virology. 2020; 127: 104370. PubMed Abstract | Publisher Full Text\n\nColling ME, Kanthi Y: COVID-19-associated coagulopathy: An exploration of mechanisms. Vascular Medicine. 2020; 25(5): 471â478. PubMed Abstract | Publisher Full Text\n\nSinghania N, Bansal S, Nimmatoori DP, et al.: Current Overview on Hypercoagulability in COVID-19. American Journal of Cardiovascular Drugs. 2020; 20(5): 393â403. PubMed Abstract | Publisher Full Text\n\nHaimei MA: Pathogenesis and Treatment Strategies of COVID-19-Related Hypercoagulant and Thrombotic Complications. Clinical and Applied Thrombosis/Hemostasis: Official Journal of the International Academy of Clinical and Applied Thrombosis/Hemostasis. 2020; 26: 107602962094449. Publisher Full Text\n\nLi H, Liu L, Zhang D, et al.: SARS-CoV-2 and viral sepsis: observations and hypotheses. The Lancet. 2020; 395(10235): 1517â1520. PubMed Abstract | Publisher Full Text\n\nWilson JG, Simpson LJ, Ferreira A-M, et al.: Cytokine profile in plasma of severe COVID-19 does not differ from ARDS and sepsis. JCI. Insight. 2020; 5(17). Publisher Full Text\n\nArslan Y, Yilmaz G, Dogan D, et al.: The effectiveness of early anticoagulant treatment in Covid-19 patients. Phlebology. 2021; 36(5): 384â391. PubMed Abstract | Publisher Full Text\n\nFlaczyk A, Rosovsky RP, Reed CT, et al.: Comparison of published guidelines for management of coagulopathy and thrombosis in critically ill patients with COVID 19: implications for clinical practice and future investigations. Critical Care. 2020; 24(1): 559. PubMed Abstract | Publisher Full Text\n\nHardy M, Lecompte T, Douxfils J, et al.: Management of the thrombotic risk associated with COVID-19: guidance for the hemostasis laboratory. Thrombosis Journal. 2020; 18(1): 17. PubMed Abstract | Publisher Full Text\n\nKarim S, Islam A, Rafiq S, et al.: The COVID-19 Pandemic: Disproportionate Thrombotic Tendency and Management Recommendations. Trop Med. Infectious Diseases. 2021; 6(1). Publisher Full Text\n\nLong X, Zhang Z, Zou W, et al.: Coagulopathy of Patients with COVID-19 is Associated with Infectious and Inflammatory Markers. Risk Management and Healthcare Policy. 2020; 13: 1965â1975. PubMed Abstract | Publisher Full Text\n\nFriedrich MS, Studt JD, Braun J, et al.: Coronavirus-induced coagulopathy during the course of disease. PLoS One. 2020; 15(12): e0243409. PubMed Abstract | Publisher Full Text\n\nWijaya I, Andhika R, Huang I: The Use of Therapeutic-Dose Anticoagulation and Its Effect on Mortality in Patients With COVID-19: A Systematic Review. Clinical and Applied Thrombosis/Hemostasis: Official Journal of the International Academy of Clinical and Applied Thrombosis/Hemostasis. 2020; 26: 107602962096079. Publisher Full Text\n\nRico-Mesa JS, Rosas D, Ahmadian-Tehrani A, et al.: The Role of Anticoagulation in COVID-19-Induced Hypercoagulability. Current Cardiology Reports. 2020; 22(7): 53. PubMed Abstract | Publisher Full Text\n\nNadkarni GN, Lala A, Bagiella E, et al.: Anticoagulation, Bleeding, Mortality, and Pathology in Hospitalized Patients With COVID-19. Journal of the American College of Cardiology. 2020; 76(16): 1815â1826. PubMed Abstract | Publisher Full Text\n\nPawlowski C, Venkatakrishnan AJ, Kirkup C, et al.: Enoxaparin is associated with lower rates of mortality than unfractionated Heparin in hospitalized COVID-19 patients. EClinicalMedicine. 2021; 33: 100774. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "126644",
"date": "09 Mar 2022",
"name": "Zhongheng Zhang",
"expertise": [
"Reviewer Expertise emergency and critical care"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting clinical question relevant to the risk stratification for COVID-19. However, I can find numerous risk stratification studies by utilizing exactly the lab measurements for the disease and there is lack of novelty in the current study. I also have several detailed comments:\n1. I do not think the discussion on anticoagulation strategy is relevant for the current study. The current study is a risk factor analysis and the causality of heparin intervention is not explored.\n2. The data analysis results described in the abstract do not support the conclusion that \"Anticoagulants in COVID-19 patients lower inflammation markers.\"; the causal inference has not been implemented formally.\n3. Many patients would be discharged/dead before day 13, how did you handle such competing risk? In such a situation, death can be regarded as competing risk if you are focusing on inflammation outcomes.\n4. \"After all the completed researches, further study on how to use anticoagulants is still being analysed to promote the best prognosis.\"---this discussion is irrelevant to the current study.\n5. I suggest that the authors should extract data on heparin use and then explore the causality between heparin and mortality, while stratified by subgroups of population.\n6. \"We also determined the relationship between inflammatory state after anti-coagulant with patientsâ mortality, along with some variables, which were age, gender, disease severity, and comorbidity.\"---this statement is confusing. You need to specify to use multivariable regression model to adjust for confounding. Furthermore, the model specification described here does allow replication of the study. For example, did you consider interaction or non-linearity for the relationship between covariates and outcome? You assumed linearity but may not hold true in real world data. You need to discuss the limitation of current study in adjusting for confounding factors. However, another approach is to use ensemble modelling1, which can address non-linearity automatically without pre-specification. At least you need to mention this in the discussion section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 2
|
https://f1000research.com/articles/11-61
|
https://f1000research.com/articles/12-338/v1
|
27 Mar 23
|
{
"type": "Review",
"title": "Exploring the anti-inflammatory activities, mechanism of action and prospective drug delivery systems of tocotrienol to target neurodegenerative diseases",
"authors": [
"Angela Maria Mathew",
"Saatheeyavaane Bhuvanendran",
"Rajesh Sreedharan Nair",
"Ammu K Radhakrishnan",
"Angela Maria Mathew",
"Rajesh Sreedharan Nair",
"Ammu K Radhakrishnan"
],
"abstract": "A major cause of death in the elderly worldwide is attributed to neurodegenerative diseases, such as AD (Alzheimerâs disease), PD (Parkinsonâs disease), ALS (Amyotrophic lateral sclerosis), FRDA (Friedreichâs ataxia), VaD (Vascular dementia) etc. These can be caused due to multiple factors such as genetic, physiological problems like stroke or tumor, or even external causes like viruses, toxins, or chemicals. T3s (tocotrienols) exhibit various bioactive properties where it acts as an antioxidant, anti-inflammatory, anti-tumorigenic, and cholesterol lowering agent. Since T3 interferes with and influences several anti-inflammatory mechanisms, it aids in combating inflammatory responses that lead to disease progression. T3s are found to have a profound neuroprotective ability, however, due to their poor oral bioavailability, their full potential could not be exploited. Hence there is a need to explore other drug delivery techniques, especially focusing on aspects of nanotechnology. In this review paper we explore the anti-inflammatory mechanisms of T3 to apply it in the treatment of neurodegenerative diseases and also discusses the possibilities of nano methods of administering tocotrienols to target neurodegenerative diseases.",
"keywords": [
"Tocotrienols",
"anti-inflammatory",
"neurodegenerative diseases",
"drug delivery",
"nanotechnology",
"vitamin E"
],
"content": "Introduction\n\nInitially known as âan anti-sterility factor, X,â vitamin E was first discovered in 1922 by Evans and Bishop, as it positively aided reproduction.1 Vitamin E consists of eight isoforms, namely, α, β, γ, ÎŽ - tocopherols and α, β, γ, ÎŽ - tocotrienols. The Tphs (tocopherols) and T3s (tocotrienols) contain a chromanol ring (bicyclic phenols) and hydrocarbon side chain, wherein Tphs have an aliphatic saturated phytyl tail and T3s have an unsaturated farnesyl tail.2\n\nLike other lipophilic vitamins, vitamin E absorption depends on a fat-rich diet, bile salts, and pancreatic enzymes. When supplemented orally, vitamin E homologs are absorbed via the mesenteric lymph nodes in the intestine as chylomicrons, a huge lipoprotein containing triglycerides composed of lipids from cholesterol and fatty acids, that either enter the tissues or the liver. The lipoprotein lipase (usually found in the adipose and muscle tissue) enzyme, bound to the endothelial cells of the capillaries, hydrolyses these vitamin E chylomicrons and aids in the transportation of vitamin E and other lipids to the tissues. Most of the vitamin E in the chylomicron remnants is assimilated into VLDL (very-low-density lipoprotein) in the liver, and the remaining is eliminated in the bile secretions. Cytochrome P450 metabolizes vitamin E through hydroxylation and oxidation of its side chains. The end product metabolites are eliminated in feces and urine, namely, CMBHC (carboxymethylbutyl hydroxychromans) and CEHC (carboxyethyl hydroxychromans).3\n\nWhile drawing a comparison between the Tphs and T3s, it can be found that the former has a better biopotency than the latter, with respect to the biological half-life (t1/2), which is in turn linked to its side chain tail length. Due to its unsaturated carbon tail, T3 is metabolized, degraded, and excreted in the urine quicker than Tph. Human cytochrome P450 (called CYP4F2) catalyzes Ï-hydroxylation, and Tph and T3 are converted to carboxychromanols. Numerous factors contribute to the degradation of vitamin E isoforms, including their respective molecular structures, the number of methyl groups attached to the chromanol ring, and the stereochemistry of the carbon tail. The intracellular metabolic breakdown of vitamin E isomers was found to be catalyzed by only the CYP4F2 enzyme (t1/2).4 Due to its unsaturated isoprenoid carbon tail, the T3 tail is shorter than Tph. This property of T3 is also found to have many superior bioactivities than Tph, which will be discussed in the following sections.\n\nThe functional aspect of T3 can provide neuroprotection against oxidative stress induced by free radicals, resulting in defective synapses and neuronal damage. The structure and functionality of the neurons were sustained upon T3 administration, which was evaluated based on their neuroprotective properties against neurotoxic metabolites involved in diminishing cognitive functions, like NO (nitric oxide) and glutamate-induced ROS (reactive oxygen species). It is found that orally supplemented T3 can enter systemic circulation and is delivered to the brain and cerebrospinal fluid by crossing the BBB (bloodâbrain barrier).4\n\nDNA chip analysis found that T3 downregulated the gene expression of PKC (protein kinase C) in HUVEC (human umbilical vein endothelial cells), exhibiting an anti-telomerase activity and downregulating VEGF (vascular endothelial growth factor) receptor in endothelial cells, thus proving its ability to inhibit angiogenesis.5 Although vitamin E isomers were found to have potential anticancer activities due to their low solubility (in aqueous media) and bioavailability when administered orally, in vivo studies in animals are often affected. Vitamin E formulations in solvents like DMSO (dimethyl sulfoxide), ethanol, and vegetable oil emulsions, also have a disadvantage in clinical trials owing to their hydrophobic nature.6\n\nIn this review, we focus on exploring the anti-inflammatory property of T3 and its application to treat progressive neurodegenerative diseases, alongside discussing the various drug delivery techniques of T3 to ensure greater bioavailability.\n\nVitamin E is a lipophilic vitamin, which has eight naturally occurring isoforms, namely, α, β, γ, ÎŽ - tocopherol and α, β, γ, ÎŽ - tocotrienol. The α-Tph has been studied the most because of its increased bioavailability from the diet, and also it is found in almost every cell in the body. Due to the presence of α-Tph, the dietary uptake of T3 homologs is usually reduced, leading to less bioavailability via oral administration. Majorly due to this reason, for many years, researchers have been focusing more on the properties of α-Tphs. Even the absorbed β, γ, ÎŽ-Tphs are eliminated through the bile secretions and finally removed via feces, whereas the α-Tph is excreted through the urine.7 But in recent years, it has been found that T3 has more potent antioxidant and anti-inflammatory properties. All these will be discussed in the later sections of this review.\n\nChemical aspect\n\nVitamin E is a hydrophobic/lipophilic phenolic compound. Vitamin E isomers, namely Tph and T3, are known as tocochromanols. They have a chromanol ring and a phytyl tail (Figure 1). T3 has a farnesyl tail with three double bonds at carbon 3, 7, and 11 and has four isoforms, α, β, γ, and ÎŽ-T3, based on the position of the methyl group on the chromanol ring. On the other hand, Tph has a saturated phytyl tail; this difference in the long carbon tail accounts for the varied biological activities of Tph and T3. The β-T3 and γ-T3 are structural isomers and contain the same number of the methyl group on the chromanol ring. In comparison to β-T3 and γ-T3, α-T3 has an extra methyl group and ÎŽ-T3 has one less methyl group (Table 1).8\n\nFunctional aspect\n\nTocotrienol has numerous pharmaceutical applications such as antioxidant,9 anti-inflammatory,10 anti-tumorigenic,11 hypolipidemic,12 stimulates an immune response,13 cardiovascular health,14 bone health15 and neuroprotection.16 The properties of T3 that can be attributed to treating neurodegenerative diseases can be its antioxidant, cholesterol-lowering, and anti-inflammatory nature.17\n\nA major contributing factor towards the pathogenesis of many diseases such as atherosclerosis18 and other cardiovascular diseases,19 rheumatoid arthritis,20 osteoporosis,21 diabetes mellitus,22 COPD (chronic obstructive pulmonary disease),23 Alzheimerâs disease,24 and cancer,25 is inflammation. The inflammatory responses are mediated by leukotrienes and prostaglandins, whose precursor is arachidonic acid.26,27 Cyclooxygenase (COX) 1 and 2 catalyze the oxidation of arachidonic acid to synthesize prostaglandin E2 (PGE2),27 which is involved in the production of cytokines.28 Leukotriene B4, another derivative of arachidonic acid catalyzed by the 5-lipoxygenase (5-LOX) enzyme, is a strong chemotactic agent.26 Pro-inflammatory cytokines have been implicated in the pathogenesis of inflammation-related diseases. Central transcription factors, such as NFκB (nuclear factor κ-B) and JAK-STAT6/3 (Janus kinase/signal transducers and activators of transcription), facilitate the gene expression of numerous pro-inflammatory cytokines.29\n\nTRF (tocotrienol rich fraction) from palm oil has prominent anti-inflammatory activity by blocking NFκB pathway activation and can selectively inhibit COX-2 gene expression. The COX-2 downregulation also resulted in the consequent suppression of PGE2 production. This work also describes that TRF can effectively inhibit LPS (lipopolysaccharides)-induced NO production, and secretion of inflammatory cytokines like IL-4, IL-8, TNF-α and iNOS (inducible nitric oxide synthase) in a dose-dependent manner in LPS-stimulated human monocytic THP1 cells.30 T3, inhibiting NFκB and mTOR (mammalian target of rapamycin) suppresses the SASP (senescence-associated secretory phenotype) produced by senescent cells or even selectively removes these aged cells through senolysis (selective lysis of senescent death).10\n\nÎŽ-T3 has a rare dual biological property, which was the first to be discovered in any naturally-occurring compound to possess this characteristic, where it has both anti-inflammatory (inhibition) and pro-inflammatory (activation) properties based on its concentration.31\n\nFor the first time, it was reported that γ-T3 could lower the inflammation and oxidative stress induced by cigarette smoking via enhancing Nrf2 (nuclear factor erythroid-2-related factor 2) activation and inhibiting the nuclear translocation of pro-inflammatory transcription factors like NFκB and STAT3.32 γ-T3 upregulates A20 (inhibitor of NFκB) and thereby inhibits the TNF-α-induced activation of NFκB via modulating the sphingolipid pathways.33\n\nA study conducted in the FeNTA (ferric nitrilotriacetate) model reported that palm-oil-derived tocotrienol is more potent in protecting against bone resorption, eventually leading to bone loss and osteoporosis. It was found that the palm-oil tocotrienol mixture significantly lowered the levels of pro-inflammatory cytokines such as IL-1 and IL-6 caused by FeNTA toxicity.34 It was also suggested that this property could be because of its antioxidant activity.\n\nÎŽ-T3 can decrease the production of pro-inflammatory cytokines like IL-6 and MCP-1 (monocyte chemoattractant protein) and consecutively increase the secretion of anti-inflammatory cytokines like IL-10, ultimately lowering inflammation and improving lipid metabolism. In addition, it also reduced liver triglycerides, macrophage infiltration, and adipocyte size, resulting in an overall better metabolically healthy profile.35 d-ÎŽ-T3 can mediate the downregulation of PPAR-γ (peroxisome proliferator-activated receptors) leading to reduced levels of triglycerides within the cells, followed by decreased uptake of glucose and lower levels of proteins such as HMG CoA (hydroxyl-methyl-glutaryl co-enzyme A) reductase, p-Akt (PKB - Protein kinase B) and GLUT-4.36 In LPS-stimulated macrophages, ÎŽ-T3 can prevent inflammation by inhibiting LPS-stimulated NO, suppressing NFκB and AP-1, and also inhibiting the phosphorylation of JNK (c-Jun N-terminal kinase) and ERK1/2 (extracellular regulated protein kinases), and proinflammatory cytokines (IL-1β, IL-6, IFN-γ, TNF-α). Altogether ÎŽ-T3 inhibits the MAPK (mitogen-activated protein kinase) and PPAR-activated signaling.37\n\nγ-T3 can delay the onset of diabetes mellitus by inhibiting NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome38 and has anti-adipogenic action via AMPK (5â² AMP-activated protein kinase) and autophagy activation.39 γ-T3 downregulates the C/EBP-β and upregulates C/EBP-α, contributing to the inhibition of adipocyte differentiation at an early phase.39 By suppressing NFκB activation, γ-T3 could attenuate the surged IL-6 and MCP-1 secretion caused by TNF-α induced inflammation. It regulates adiponectin secretion via PPAR-γ gene expression in adipocytes.40 The suppression of NFκB signaling by T3 isomers leads to the halting of tissue inflammation and can be credited to its anti-inflammatory property.\n\nAnother important aspect that needs to be addressed is âwhether the anti-inflammatory and anti-oxidant properties go hand-in-hand?â. The antioxidant and anti-inflammatory ability of T3 are intrinsically linked through numerous mechanisms since inflammatory response accompanies oxidative stress.41 Chronic tissue inflammation can be accounted for by the oxidative stress produced within cells due to cellular damage caused by ROS. Both antioxidant and anti-inflammatory signaling pathways complement each other since antioxidants alleviate oxidative damage by scavenging free radicals. A bioactive compound with an antioxidant effect will undoubtedly be an anti-inflammatory. Vitamin E homologs are popular dietary supplements, especially due to their antioxidant activities, wherein they prevent inflammatory responses in the cellular microenvironment caused by endogenous and exogenous oxidative agents. T3, a non-enzymatic antioxidant compound, can influence the enzymatic counterparts, including enzymes like SOD (super oxide dismutase), glutathione oxidase, CAT (catalase), peroxiredoxin and redox proteins, heme oxygenase-1, and glutathione transferase.4 In vitamin E isoforms, the antioxidant property is based on the number of hydroxyl groups, and the T3 has a superior antioxidant property than Tph, which is in the order α > β > γ > ÎŽ, and can be credited to their greater distribution in the plasma membrane bilayer and interaction with lipid peroxyl radicals.2 Figure 2 and Figure 3 demonstrate the anti-inflammatory properties of T3.\n\nAbbreviations: IL - Interleukin, MCP - Monocyte chemoattractant protein, TNF - Tumor necrosis factor, NFκB - Nuclear factor κ-B, IFN - Interferon, MAPK - Mitogen-activated protein kinase, PPAR - Peroxisome proliferator-activated receptors, mTOR - Mammalian target of rapamycin, LPS - Lipopolysaccharide, NO - Nitric oxide, COX - Cyclooxygenase, AP - Activator protein, Nrf2 - nuclear factor erythroid-2-related factor 2, HMG Co A - hydroxyl-methyl-glutaryl co-enzyme A.\n\nAbbreviations: LPS - Lipopolysaccharide, NO - Nitric oxide, TNF - Tumor necrosis factor, COX - Cyclooxygenase, LOX - Lipoxygenase, NFκB - Nuclear factor κ-B, iNOS - Inducible nitric oxide synthase, AP - Activator protein, PGE2 - Prostaglandin E2, IL - Interleukin, Nrf2 - nuclear factor erythroid-2-related factor 2, IFN - Interferon, SOD - Super oxide dismutase, CAT - Catalase, ROS - Reactive oxygen species, HMG Co A - hydroxyl-methyl-glutaryl co-enzyme A, SASP - senescence-associated secretory phenotype, JAK/STAT - Janus kinase/signal transducers and activators of transcription, MCP - Monocyte chemoattractant protein, NLRP3 - NOD-, LRR- and pyrin domain-containing protein 3, AMPK - 5' AMP-activated protein kinase, PPAR - Peroxisome proliferator-activated receptors, JNK - c-Jun N-terminal kinase, ERK - Extracellular regulated protein kinases, MAPK - Mitogen-activated protein kinase, mTOR - Mammalian target of rapamycin, c-Src â cellular Src, MMP - Matrix metalloproteinase, Iba - Ionized calcium-binding adapter molecule.\n\nT3 has superior biological properties than Tph, with prominent antioxidant and anti-inflammatory effects. T3 can hinder the mevalonate pathway suppressing cholesterol biosynthesis.42 Hepatic HMG CoA reductase inhibition can be attributed to an unsaturated hydrocarbon side chain.43 T3 downregulates the HMG Co-A reductase due to this hypolipidemic and anti-inflammatory ability implicated in the medical condition of AD.44\n\nThe tissue-specific macrophages called the microglia are involved in the neurogenesis and homeostasis in the CNS, which react to various stimuli via the secretion of inflammatory cytokines. These inflammatory cytokines can be considered a warning sign for identifying the occurrence of neuro-diseases like AD, PD, ALS, stroke, multiple sclerosis, etc. ÎŽ-T3 derived from palm oil attenuates NO production, IL-1β expression, and 5-LOX mRNA expression in BV2 microglia. It also inhibited the expression of PGE2 and selective inhibition of COX-2 and not COX-1.45 The neuroprotective effects of various T3s are listed out in Table 2.\n\nAbbreviations: ROS - Reactive oxygen species, Aβ - Amyloid β, SOD - Superoxide dismutase, CAT - Catalase, F2-IsoPs - F2-isoPs - F2-isoprostanes, SN - Substantia nigra, STR - Striatum, TH - Tyrosine hydroxylase, TNF - Tumor necrosis factor, MCP - Monocyte chemoattractant protein, MMP - Matrix metalloproteinase, Iba - Ionized calcium-binding adapter molecule, CMR - Cardiac magnetic resonance, PDGF-C - Platelet-derived growth factor-C.\n\nAD\n\nAD is a neurodegenerative illness that is manifested as a progressive dementia leading to cognitive and memory impairment and various other biological changes such as Aβ (amyloid β) plaque accumulation, neurofibrillary entanglement, synaptic loss, to name a few. High cholesterol levels are linked to the pathogenesis of AD. A surge in cholesterol levels and cholesterol biosynthesis will lead to proteolytic cleavage of APP (amyloid precursor protein) by β-secretase and γ-secretase to produce Aβ.46\n\nThe anti-inflammatory activity of T3 can be attributed to its protection against AD.47 TRF could successfully hinder the progression of AD by regulating multiple genes and signaling pathways involved in the pathogenesis.48 Ibrahim et al.49 found that when administered at higher concentrations, α-Tph was able to decrease the Aβ aggregation, whereas, α-T3 even at lower concentrations was able to both reduce the Aβ aggregation and disaggregate the already formed Aβ fibrils. γ-T3, on the other hand, exhibited the ability to reduce the Aβ oligomerization, apart from lowering the Aβ aggregation and disaggregation of Aβ fibrils.49\n\nAlongside the positive effects of T3, it is also found to have some negative impacts on AD. α-Tph and α-T3 tend to enhance the production of Aβ (via β-secretase and γ-secretase production) and decrease the degradation of Aβ, thereby facilitating the accumulation of plaques.50 When vitamin E alone had the potential to increase p-Akt, lower oxidative stress via decreasing ROS production that occurs as a result of insulin resistance, and reduce AD markers like GSK3β and TAU. Vitamin E, in combination with vitamin D, showed a notable increase in GLUT4, p-Akt, and reduced ROS and AD markers.51\n\nPD\n\nPD is a progressive neurodegenerative disease, whose pathological conditions can be attributed to the loss of dopaminergic neurons in the midbrain and also to the neuroinflammation caused by neuroglial activation, with clinical abnormalities including motor and non-motor symptoms. A recent study found that both α-T3 and γ-T3 can alleviate neuronal damage caused by 6-OHDA and provide neuroprotection, but it was α-T3 that had a better potential to reduce neuroinflammation.16 The studies conducted by Nakaso et al.52 found that γ-T3 and ÎŽ-T3 are involved in the activation of PI3K/Akt signaling by binding to Erβ (estrogen receptors) leading to caveola formation when the SH-SY5Y neural cells were subjected to MPP+ toxicity. They have also stated that the antioxidant property of T3 is not responsible for this effect. Later Nakaso et al.53 reported that ÎŽ-T3 increased motor activities and prevented the loss of neurons in the SN when conducted experiments in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced C57BL/6 mice. Currently there is an ongoing randomized clinical trial in phase II (NCT04491383), to explore the potential of T3 to slow down the progression of motor and non-motor dysfunctions caused by PD, which was based on previously proven results from experimentation on transgenic animals. This study is being conducted in 100 PD patients who are orally administered with T3 (Tocovid Suprabio (HOV-12020)) or placebo for 104 weeks.54\n\nStroke\n\nAn in vitro study using cultured HT4 hippocampal nerve cells found that, unlike α-Tph, even the nanomolar concentration of α-T3 could suppress the early activation of glutamate-induced c-Src (cellular Src) kinase, by especially blocking glutamate-induced death and thereby saving the neurons.55 Complementing this discovery, another research proved that T3 acts as an important checkpoint to protect against neurodegeneration caused by glutamate-induced cell death and stroke.56\n\nFRDA\n\nAnother neurodegenerative disease Friedreichâs ataxia (FRDA) is caused due to the downregulation of frataxin, which is a mitochondrial protein. Studies claim that FRDA patients can encounter ferroptosis (caused by impaired mitochondrial failure, lipid peroxidation and glutathione metabolism ultimately leading to iron-dependent cell death). LOX inhibitors such as Tph or T3 [EPI-743 (α-tocotrienol quinone which is now known as vatiquinone or PTC-743) and SFN (sulforaphane) are also Nrf2 inducers], could prevent ferroptosis. Upon study conducted on mouse myoblast with frataxin-gene silenced, cardiac cells of frataxin-KIKO (knockin/knockout) mouse model, fibroblasts of skin and blood of human patients, it was concluded that Nrf2 inducers could neutralize the ferroptosis and that ferroptosis has close correlation with Nrf2 activation.57 EPI-743 is a therapeutic that targets oxidoreductases involved in redox mechanisms. In a randomized placebo-controlled clinical trial (NCT01728064) to determine the clinical effects and safety of EPI-743 in FA patients, it was found that EPI-743 was well tolerated and safe for administration and also that after 24 months of treatment with this drug, a prominent long-term improvement was observed in the neurological function and disease progression.58 Another study performed with five patients who were supplemented a T3 mixture and idebenone found that these patients showed normal results for oxidative stress parameters and other inflammation indices. In addition, they also exhibited an improvement in CMR (cardiac magnetic resonance) readings suggesting a reduction in myocardial damage.59\n\nVaD\n\nVaD is the second most common form of dementia, which can be characterized as a gradual drop of cognition. Type 2 diabetes (T2D) patients are at high risk of getting this disease. In the study conducted in T2D rat models, it was concluded that treatment using TRF prevented memory loss and attenuated various biochemical parameters like plasma homocysteine (HCY), blood glucose, SOD levels, acetylcholinesterase (AChE), glutathione (GSH), immunohistochemistry for platelet-derived growth factor-C (PDGF-C).60\n\nBioavailability\n\nα-TTP (α-tocopherol transfer protein), found in the hepatocytic cytosol, binds the vitamin E administered orally and emphasizes its effective transport between the membranes, identified in rats61 and humans.62 RRR-α-tocopherol is the most biologically active and naturally occurring stereoisomer of α-tocopherol.63 The α-TTP has a higher affinity to this isomer due to the affinity of α-TTP towards the forms of R,64 thus resulting in discrimination. Studies confirmed the existence of bio-discrimination against T3s due to this affinity.65 The absorption of other vitamin E isoforms like γ-T3, if supplemented orally along with α-Tph, will face hindrance because α-TTP binds the α-Tph selectively.66\n\nStudies have shown that T3 has excellent antioxidant activity in in vivo systems, but its oral bioavailability is low to rare since they are not identified by the α-TTP.67 According to in vivo studies, the plasma concentration of T3 is much lower in the presence of α-Tph. The short t1/2 of T3 adds up to its poor bioavailability.68\n\nThe highly unsaturated isoprenoid tail contributes to the ability of T3 isomers to cross lipid layers of the brain, liver, and adipose tissue efficiently. γ-T3 in GDT (gamma delta tocotrienol) has been found to have a better bioavailability than TRF, while ÎŽ-T3 had a lower bioavailability. A significantly high γ-T3 concentration was found localized in the adipose tissue.69\n\nVitamin E is absorbed via two mechanisms, primarily in the distal portions of the small intestine across the apical enterocyte membrane by passive transport, also assisted by SR-B1, CD36, and NPC1L1 (Niemann-Pick C1-like 1), which are vitamin D transporters. Secondly, through the lymphatic system, as a lipoprotein complex that is composed of bile salt micelles, vitamin E, and chylomicron. Due to its huge size, lipoprotein cannot be transported through the blood capillaries.70\n\nA comparative study of bioavailability and intestinal absorption kinetics, of γ-T3 and α-Tph, both in vitro interaction kinetics (in aqueous media) and in vivo (in oil media) in rats demonstrated that α-Tph was found to have a significant oral bioavailability (36%) than γ-T3 (9%), which in turn can be concluded based on in situ studies, that the α-Tph has a strong intestinal permeability compared to γ-T3. The findings of this study suggest that γ-T3 uptake and bioavailability can be improved by enhancing the solubility and dissolution of γ-T3, leading to its increased permeability. This can be attained either by structural modifications or by exploring new formulation approaches.71\n\nA study conducted in α-TTP-deficient mice proved that α-T3 supplemented orally could restore fertility, and a greater amount of this vitamin was transferred to vital organs.55 Another study demonstrates that α-TTP gene expression in the cultured hepatocytic cells was not substantially necessary for the intracellular localization of α-Tph, γ-Tph, α-T3, and γ-T3. It was also found that neither the methylation of the chromanol ring nor the side chain saturation had any significant role in the distribution of these vitamin E isomers within the cells. These results further indicate the possibility of passive transport, like diffusion, as the key driving force.72 Hence, there could be other mechanisms apart from α-TTP governing the uptake of T3 into the cells.\n\nOther parenteral vitamin E administration modes include intraperitoneal, intravenous, intramuscular, and subcutaneous injections.3\n\nAn important strategy to overcome the lower bioavailability of compounds is to opt for nanomedicine, wherein we can take advantage of the small size and greater surface area for effective drug delivery. The preclinical development of T3 nanocarriers comprises niosomes, polymeric NPs (nanoparticles), NEs (nanoemulsions), and NLCs (nanostructured lipid carriers).73 The nanoformulations of hydrophobic/lipophilic compounds or drugs ensure several applications like protection from degradation and enhanced absorption in the GIT (gastrointestinal tract), extended systemic circulation, and regulated drug release.3 Moreover, this technique overcomes the side effects of chemotherapeutic drugs, such as systemic toxicity, and favors accumulation in the specific tumor-infected tissue via target-specific markers to narrow down the possibilities of off-target dispersal.74 It can also improve the half-life, chemical integrity, solubility, and membrane permeability. The interaction of vitamin E with other lipophilic vitamins often leads to its hindered uptake, whereas nanoparticles encapsulating vitamin E can overcome this hurdle.70\n\nNPs usually range in size from 1â100 nm, wherein they either contain drugs distributed evenly within the nanospheres matrix, or a polymer membrane entraps the drug in its cavity (nanocapsules). Novel drug delivery systems are being explored to improvise the pharmacokinetics, bioavailability, stability, and target-specificity of vitamin E, simultaneously by minimizing side effects.6\n\nThe encapsulation efficiency of various nanoparticulate systems depends on factors such as the structure of the lipid matrix with respect to crystallinity and the nature of the compound (hydrophilic or lipophilic) to decide lipid compatibility. Since vitamin E is lipophilic, it is compatible with the lipid matrix, assuring higher encapsulation efficiency (EE) and drug loading. These characteristics are quantified to determine the amount of NPs based on the purpose, target, and drug release. Due to its lipophilic nature, vitamin E can be retained in the lipid phase during the NP formation and the majority of the studies claims that it had enhanced EE% (>70%), and even few among them reported values nearly equal to 100%.75\n\nPLGA-Chi (poly-lactic-co-glycolic acid - chitosan) NPs (about 3.5 folds) observed a potentially greater cellular uptake compared to PLGA NPs (both NPs encapsulated with α-Tph and TRF). Both these NPs were able to exhibit antioxidant and antiproliferative properties, and this method can assure greater bioavailability and exploit the pharmaceutical potential of hydrophobic compounds like T3 and Tphs, due to their entrapment abilities.76\n\nSelf-emulsifying drug delivery systems (SEDDS)\n\nA colloidal dispersion of T3 can be enhanced using SEDDS, which consists of oils, surfactants, and co-solvent. Once introduced into the GIT, the T3 SEDDS becomes a nanoemulsion (100â 300 nm in size) in the presence of gastrointestinal fluids, facilitating in situ solubilization of T3. This solubilization aids in the greater surface area for drug release and improves drug partitioning across the enterocytic membrane, thus surpassing the first-pass metabolism via entering the lymphatic system.77\n\nWhen administered orally, the uptake of T3 becomes limited due to low bioavailability, with <30% of α-T3 and <10% of γ-T3 and ÎŽ-T3.78 Due to its lipophilic nature, T3 has poor solubility in the aqueous medium and is a perfect candidate for s-SEDDS to enhance oral bioavailability. These formulations include oils and water-insoluble surfactants, which form fine emulsions or self-emulsions upon exposure to aqueous media and distribute readily in the gastrointestinal tract until it's absorbed.79\n\nIn vivo study performed in SD rats using s-SEDDS (solid self-emulsifying drug delivery systems) containing 70%, TRF has been shown to effectively ensure better oral bioavailability of α, γ, and ÎŽ-T3, with almost similar bioavailability. When the s-SEDDS T3 powder was administered in combination with two different surfactants, namely poloxamer and Labrasol®, this formulation exhibited a faster absorption rate than that of formulations with only one of these surfactants and also non-self-emulsifying liquid TRF.80\n\nStudies have shown that by incorporating γ-T3 in the formulation of a self-emulsifying drug delivery system (SEDDS), its solubilization and passive permeability were significantly enhanced, therefore, improving its oral bioavailability and intracellular absorption compared to the Tocovid (a commercially available form of T3).81\n\nγ-T3, with its unsaturated phytyl chain, can easily enter tissues, ultimately resulting in an even distribution in the lipid bilayer of the plasma membrane. Its tissue uptake is assumed to be facilitated by receptor-mediated lipoprotein endocytosis and aided by lipoprotein lipases. The γ-T3 evenly distributed in the membrane interacts with lipid radicals using its chromanol head, readily eliminating peroxyl radicals.82\n\nLipid-based nanoencapsulation methods\n\nNanoencapsulation is a method to improve bioavailability where a matrix (secondary or shell material) entraps or encapsulates bioactive core materials, forming nanocapsules together. This drug delivery technique can be implemented to deliver oil-phase or water-phase successfully, prepared using emulsifiers, co-solvents, and carrier substances. The core material includes vitamins, minerals, lipids, enzymes, antioxidants (e.g. terpene, black pepper, and limonene), probiotics (such as Lactobacillus, and Bifidobacteria), etc. The matrix is usually constituted by lipids, cellulose, gum (gum acacia, gum arabic), etc. Modified polysaccharides are being used in the nanoencapsulation process of target molecules, assisted by various fabrication techniques like supercritical fluid, electrospray, spray drying, reverse micelle, electrospinning, coacervation, etc.83\n\nLipid-based nanoencapsulation has low toxicity, greater encapsulation efficiency (EE%), and can be produced at an industrial scale. Owing to the amphiphilic nature of these NPs, this technique also serves the advantage of encapsulating hydrophilic and lipophilic compounds with different polarities simultaneously, such as polyphenols, flavonoids, fatty acids, and carotenoids.84\n\nLiposomes and nanoliposomes\n\nThe sub-micron bilayer lipid vesicles or nanoliposomes are colloidal vesicles that entrap bioactive compounds and deliver them to the target site, enhancing bioavailability, reducing toxicity, and side effects, controlling the release of the drug, extending shelf life, etc.83\n\nCurcumin extracted from the plant Curcuma longa is a lipophilic phytochemical with numerous bioactive properties such as antioxidant, antimicrobial, anti-inflammatory, and anti-tumorigenic. Even though curcumin has the least oral toxicity and highest bioactivities compared to its curcuminoid counterparts, its hydrophobic nature, and poor water solubility results in reduced oral bioavailability.85 The curcumin-loaded nanoliposomes were found to have improved stability, physicochemical properties, controlled drug release properties, and equivalent cellular antioxidant activity (CAA). But the curcumin entrapped in nanoliposomes had lower cellular uptake than the free curcumin.86 It was found that with surface modifications of liposomes, they can be directed to treat neuro-diseases as possible phytochemical carriers. The curcumin-loaded liposome conjugated with sialic acid and wheat germ agglutinin exhibited an enhanced encapsulation efficiency and permeability through the monolayered endothelial cells in the BBB.87\n\nNanoemulsion\n\nNanoemulsions (NE) are formed by two immiscible phases (oil and water, either o/w or w/o) in the presence of one or more surfactants. These kinetically stable dispersions can be prepared through various techniques like microfluidization, high-pressure homogenization, and phase inversion temperature emulsification (PIT). These recent methods have overcome the drawbacks posed by conventional modes, such as sedimentation and creaming, producing NE with prolonged colloidal stability and low viscosity. High-pressure homogenization aided in the characterization of vitamin E nanoemulsions, wherein the lipids were digested rapidly, ensuring improved bioaccessibility, due to their smaller droplet size (80â300 nm).3 The characteristics that influence the performance of NE are zeta potential, droplet size, and drug content.88 NE approach has been used recently for controlled drug release of biologically active compounds using oil, water, and surfactants to give a colloidal nanosized dispersion. This NE system has many advantages, including increased emulsion stability, bioavailability, antioxidant effects, and altered texture. There are several NEs, namely, pickering NE, single NE, double NE, and structural NE (with the interfacial layer being single or double).83\n\nEncapsulation within a carrier increases the brainâs uptake of curcumin. For example, curcumin NE89 and curcumin-docosahexaenoic acid microemulsion (ME) enhanced the impact of the drug on malignant glioblastoma neural cells U-87.74 This method can ensure the uptake of drugs via lymphatic transportation, thus increasing the bioavailability of hydrophobic drugs.\n\nSolid lipid nanoparticles (SLN)\n\nSLNs are nanocolloidal drug delivery systems (50â1000 nm) composed of lipids, drugs, and surfactants in specific proportions. These are spherical solid structures that remain intact at room and body temperatures. It involves surfactants to stabilize the solid lipid cores. The core materials include fatty acids, acylglycerol, waxes, etc. while stabilizers involve cholesterols, phospholipids, and sphingomyelins. SLNs are used as carriers for drug encapsulation, to successfully deliver antioxidants like quercetin, curcumin, bixin, puerarin, etc. for therapeutic purposes.83\n\nThese nanostructures have several benefits, including prolonged stability, site-specific targeting, shielding labile drugs, controlled drug release, transporting both hydrophilic and lipophilic compounds, non-toxicity, ease of preparation, and cost-effectiveness. Contrary to this, SLNs pose a few drawbacks, such as a moderate drug expulsion rate due to their crystallization (under storage conditions) and limited drug loading ability.74 A hydrophobic drug, indirubin, used in ancient Chinese medicine, was loaded in SLNs to target U87MG human glioblastoma cells and was found to have enhanced the anticancer activity of this lipophilic compound.90\n\nNanostructured lipid carriers (NLCs)\n\nNLCs are second-generation lipid-based nanocarriers designed to overcome the limitations of SLNs. The NLCs can carry lipophilic and hydrophilic compounds, be oriented to site-specific targets, surface modified, controlled drug release, and have poor in vivo toxicity. Since liquid lipids are present in the nanoformulation of the NLCs, it prevented lipid crystallization that greatly decreased drug expulsion, which earlier challenged their storage.74 Additionally, it has improved drug loading capacity74 and shields the bioactive compounds from enzymatic and biological degradation. Lipid-based nanocarriers involving solid lipids mixed with liquid lipids are usually prepared by mixing both solid and liquid lipids in a ratio of 70:30.83\n\nNLCs can improve absorption through the GIT, surpassing hepatic first-pass metabolism, by entering into the mesenteric lymphatic system via M cells. P-gp (P-glycoprotein) efflux inhibition by the surfactants present on the shell of NLCs facilitates their uptake. Some lipophilic bioactive can be added to enhance GI transport. The bioavailability of hydrophobic molecule coenzyme Q10 was improved using NLC as the carrier.70 Baicalein-loaded tocol NLCs (tocol NLCs are composed of vitamin E, phospholipids, tripalmitin, gelucires, and poloxamer 188) was administered intravenously, and the vitamin E in the NLC contributed to its in vivo stability with prolonged half-life and enhanced the brain penetrating efficiency of baicalein. Profound amounts of baicalein were found in various parts of the brain, especially the cortex, brain stem, hippocampus, thalamus, striatum, and olfactory tract, when NLC was used as a carrier.91\n\nQuantum dots\n\nNanotheranostics, a technique used for therapeutic and diagnostic purposes, might overcome the challenges BBB poses. This method focuses on drug delivery systems based on quantum dots (QDs), which are zero-dimensional semiconductor nanocrystals (2â10 nm). QDs serve the advantage of having the ability to cross BBB and, due to their biocompatibility towards neurons, have low toxicity and can be oriented to target neurodegenerative diseases. Its intermediary size between discrete molecules and bulk semiconductors increases its efficiency in diagnosing and treating, mainly owing to its remarkable electrochemical and optical properties. These nanostructures possess certain distinguishing characteristics such as electronic properties, photostability, luminescence, size-tunable emission, high excitation capacity, and others, which conserve their capabilities for diagnosis, therapeutics, biosensing, and bioimaging.92 Curcumin QDs exhibited a greater potential in degrading bacteria biofilms than curcumin alone. Few QDs, like chlorophyllin and folic acid, are found to be potential candidates for imaging diagnosis.93 This opens possibilities for targeting T3 as QD to treat neurodegenerative diseases.\n\nChallenges while targeting the brain\n\nThe oral administration usually requires a heavy dosage due to systemic circulation and hepatic first-pass metabolism. Recent studies have shown that the nasal-brain route can be focused to deliver nanocarriers for drug administration as it can bypass the BBB and this may be a potential route if some of the drawbacks, such as rapid mucociliary clearance, enzymatic degradation, and low permeation rate of drug through the nasal epithelium are addressed. The CNS has barriers like BBB and BCSFB (Blood cerebrospinal fluid barrier) to shield it from the entry of neurotoxic xenobiotics and peripheral blood circulation. The BBB includes tight junctions, multidrug efflux pumps, protein transporters, enzymatic barriers, and others, while BCSFB includes tight junctions joining the choroid plexus epithelium. The physicochemical nature of the compound, such as lipophilicity and molecular weight, are the key factors influencing the transport via BBB.94\n\nThe intranasal route is non-invasive, has quick therapeutic delivery, and surpasses the first-pass metabolism and the BBB.94 Several in vivo studies prove that drug administration through the nasal epithelium can induce a better impact than any parenteral delivery method, including intravenous. Due to its mucoadhesive and penetration-enhancing ability, chitosan when incorporated into NEs, has exhibited high permeation and flux via the nasal membrane, compared to the NEs devoid of chitosan. The NEs gained an inherent mucoadhesive ability when formulated with chitosan.88\n\nOn intranasal (IN) administration, the drugâs nanodroplets are transported through the endothelium in the brain via endocytosis or transcytosis mechanisms. The drug penetrating ability is amplified by the surfactants contained in the NE, which has a fluidizing impact on the endothelium.88 The contact time in the nasal epithelium can be prolonged by inhibiting the P-gp efflux pump, which ultimately increases the intranasal administration to the brain.95\n\nIntranasal supplementation of selegiline (lipophilic) NE enhances its bioavailability to the brain due to safe-guarding from degrading enzymes in the nasal epithelium, bypassing the first-pass metabolism and systemic circulation. After this intranasal treatment in rats, it almost normalized dopamine levels along with restoration of non-enzymatic and enzymatic markers of lipid peroxidation and reverted the haloperidol effect leading to regaining the cellular integrity. Thus, intranasal delivery of selegiline was more promising since it can mend dopamine levels and antioxidant scarcity and target the CNS without crossing the BBB.96\n\nA curcumin-loaded NLC prepared using the hot high-pressure homogenization method was found to have anticancer effects and enhanced drug entrapment and release properties. X-ray diffraction (XRD) and Differential scanning calorimetry (DSC) analysis revealed that the NLCs contained the drug in an amorphous form. Following this drug administration, there was a noticeable increase of curcumin in the CNS, and also the NLC exhibited very low toxicity towards astrocytoma-glioblastoma cells and is biocompatible and biodegradable. Thus, this study concludes that IN mode of drug delivery can be an alternative route for therapeutic applications to treat neuro-diseases, including glioblastoma.97\n\n\nFuture prospects\n\nRecently, intranasal (IN) drug administration has been of particular interest to researchers since it is non-invasive, ensures greater bioavailability and offers options for a low dosage of drugs. Even though this route has many advantages, its challenges are quite difficult to overcome. More studies are to be conducted to understand the mechanism, bioavailability, uptake, and therapeutic action of T3 when targeting the brain via this route. Since nanomedicine can enhance the bioavailability of the drug, there is a wider possibility to explore the various nanocarrier methods for effectively transporting T3 to treat neurodegenerative diseases. Different combinations of lipid carriers, surfactants, and mucoadhesive polymers can be chosen based on the targeted disease condition. Since few phytochemicals when used as quantum dot candidates exhibited amplified bioactive properties, this opens possibilities to explore similar strategies for tocotrienol as a worthy candidate. If tocotrienol QD is quantified and characterized, then it can be directed toward targeting neurodegenerative diseases, which could aid in early diagnosis and treatment.\n\n\nConclusions\n\nDespite being a promising candidate with numerous therapeutic applications, T3 could not be exploited to its full potential owing to its poor oral bioavailability. Its anti-inflammatory property is of particular interest since it can be used to target progressive neurodegenerative diseases, which are usually fatal. New possible drug delivery techniques based on nanotechnology offer options to target T3 towards the brain to attenuate neurodegenerative diseases. But the characterization and quantification of T3 nanocarriers targeting the brain are yet to be proven. Successful nano-drug delivery methods of other lipophilic bioactive compounds have been discussed in this review to highlight the possibility of using similar formulations for T3. The hepatic first-pass metabolism and BBB pose serious threats to focusing the CNS (central nervous system) to treat neuro-diseases. The nose-brain route rules out this troubleshooting and allows for low dosage and lesser side effects. Intranasal administration is a promising method for T3 due to its lipophilicity and low molecular weight, more research can be focused in this arena.",
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Publisher Full Text\n\nKırdar S, Serter M, Ceylan E, et al.: Adiponectin as a biomarker of systemic inflammatory response in smoker patients with stable and exacerbation phases of chronic obstructive pulmonary disease. Scand. J. Clin. Lab. 2009; 69(2): 219â224. PubMed Abstract | Publisher Full Text\n\nAisen P: Inflammation and Alzheimerâs Disease: Mechanisms and Therapeutic Strategies. Gerontology. 1997; 43(1-2): 143â149. Publisher Full Text\n\nCoussens LM, Werb Z: Inflammation and cancer. Nature. 2002; 420(6917): 860â867. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYokomizo T, Izumi T, Shimizu T: Leukotriene B4: metabolism and signal transduction. Arch. Biochem. Biophys. 2001; 385(2): 231â241. Publisher Full Text\n\nVane JR: Prostaglandins as mediators of inflammation. Adv. Prostaglandin Thromboxane Res. 1976; 2: 791â801. PubMed Abstract\n\nWilliams JA, Shacter E: Regulation of macrophage cytokine production by prostaglandin E2. Distinct roles of cyclooxygenase-1 and -2. J. Biol. Chem. 1997; 272(41): 25693â25699. Publisher Full Text\n\nJiang Q: Natural forms of vitamin E: metabolism, antioxidant, and anti-inflammatory activities and their role in disease prevention and therapy. Free Radic. Biol. Med. 2014; 72: 76â90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu S, Liu P, Ng L: Tocotrienol-rich fraction of palm oil exhibits anti-inflammatory property by suppressing the expression of inflammatory mediators in human monocytic cells. Mol. Nutr. Food Res. 2008; 52(8): 921â929. PubMed Abstract | Publisher Full Text\n\nQureshi AA, Khan DA, Silswal N, et al.: Evaluation of Pharmacokinetics, and Bioavailability of Higher Doses of Tocotrienols in Healthy Fed Humans. J. Clin. Exp. Cardiol. 2016; 07(4): 434. Publisher Full Text\n\nPeh H, Tan W, Chan T, et al.: Vitamin E isoform γ-tocotrienol protects against emphysema in cigarette smoke-induced COPD. Free Radic. Biol. Med. 2017; 110: 332â344. PubMed Abstract | Publisher Full Text\n\nWang Y, Park N, Jang Y, et al.: Vitamin E γ-Tocotrienol Inhibits Cytokine-Stimulated NF-κB Activation by Induction of Anti-Inflammatory A20 via Stress Adaptive Response Due to Modulation of Sphingolipids. J. Immunol. 2015; 195(1): 126â133. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAhmad N, Khalid B, Luke D, et al.: TOCOTRIENOL OFFERS BETTER PROTECTION THAN TOCOPHEROL FROM FREE RADICAL-INDUCED DAMAGE OF RAT BONE. Clin. Exp. Pharmacol. Physiol. 2005; 32(9): 761â770. PubMed Abstract | Publisher Full Text\n\nAllen L, Ramalingam L, Menikdiwela K, et al.: Effects of delta-tocotrienol on obesity-related adipocyte hypertrophy, inflammation and hepatic steatosis in high-fat-fed mice. J. Nutr. Biochem. 2017; 48: 128â137. PubMed Abstract | Publisher Full Text\n\nTorabi S, Yeganehjoo H, Shen C, et al.: Peroxisome proliferatorâactivated receptor γ down-regulation mediates the inhibitory effect of d-ÎŽ-tocotrienol on the differentiation of murine 3T3-F442A preadipocytes. Nutr. Res. 2016; 36(12): 1345â1352. PubMed Abstract | Publisher Full Text\n\nShen J, Yang T, Xu Y, et al.: Î-tocotrienol, isolated from rice bran, exerts an anti-inflammatory effect via MAPKS and ppars signaling pathways in lipopolysaccharide-stimulated macrophages. Int. J. Mol. Sci. 2018; 19(10): 3022. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim Y, Wang W, Okla M, et al.: Suppression of NLRP3 inflammasome by γ-tocotrienol ameliorates type 2 diabetes. J. Lipid Res. 2016; 57(1): 66â76. Publisher Full Text\n\nZhao L, Ha J, Okla M, et al.: Activation of autophagy and AMPK by gamma-tocotrienol suppresses the adipogenesis in human adipose derived stem cells. Mol. Nutr. Food Res. 2013; 58(3): 569â579. PubMed Abstract | Publisher Full Text\n\nMatsunaga T, Shoji A, Gu N, et al.: γ-tocotrienol attenuates TNF-α-induced changes in secretion and gene expression of MCP-1, IL-6 and adiponectin in 3T3-L1 adipocytes. Mol. Med. Rep. 2012; 5: 905â909. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimpson D, Oliver P: ROS Generation in Microglia: Understanding Oxidative Stress and Inflammation in Neurodegenerative Disease. Antioxidants. 2020; 9(8): 743. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaederstorff D, Elste V, Aebischer C, et al.: Effect of either gamma-tocotrienol or a tocotrienol mixture on the plasma lipid profile in Hamsters. Ann. Nutr. Metab. 2002; 46(1): 17â23. Publisher Full Text\n\nQureshi AA, Reis JC, Papasian CJ, et al.: Tocotrienols inhibit lipopolysaccharide-induced pro-inflammatory cytokines in macrophages of female mice. Lipids Health Dis. 2010; 9(1): 143. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrowne D, McGuinness B, Woodside J, et al.: Vitamin E and Alzheimerâs disease: what do we know so far? Clin. Interv. Aging. 2019; 14: 1303â1317. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan S, Israf Ali D, Khaza'ai H, et al.: Cellular uptake and anti-inflammatory effects of palm oil-derived delta (ÎŽ)-tocotrienol in microglia. Cell. Immunol. 2020; 357: 104200. Publisher Full Text\n\nChin K, Tay S: A Review on the Relationship between Tocotrienol and Alzheimer Disease. Nutrients. 2018; 10(7): 881. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaomi R, Shafie NH, Kaniappan P, et al.: An interactive review on the role of Tocotrienols in the neurodegenerative disorders. Front. Nutr. 2021; 8: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWan Nasri W, Makpol S, Mazlan M, et al.: Tocotrienol Rich Fraction Supplementation Modulate Brain Hippocampal Gene Expression in APPswe/PS1dE9 Alzheimerâs Disease Mouse Model. J. Alzheimers Dis. 2019; 70(s1): S239âS254. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIbrahim NF, Hamezah HS, Yanagisawa D, et al.: The effect of α-tocopherol, α- and γ-tocotrienols on amyloid-β aggregation and disaggregation in vitro. Biochem. Biophys. Rep. 2021; 28: 101131. Publisher Full Text\n\nGrimm M, Regner L, Mett J, et al.: Tocotrienol Affects Oxidative Stress, Cholesterol Homeostasis and the Amyloidogenic Pathway in Neuroblastoma Cells: Consequences for Alzheimerâs Disease. Int. J. Mol. Sci. 2016; 17(11): 1809. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaulkffali A, Md Razip N, Syed Alwi S, et al.: Vitamins D and E Stimulate the PI3K-AKT Signalling Pathway in Insulin-Resistant SK-N-SH Neuronal Cells. Nutrients. 2019; 11(10): 2525. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakaso K, Tajima N, Horikoshi Y, et al.: The estrogen receptor β-PI3K/Akt pathway mediates the cytoprotective effects of tocotrienol in a cellular Parkinson's disease model. Biochim. Biophys. Acta Mol. Basis Dis. 2014; 1842(9): 1303â1312. PubMed Abstract | Publisher Full Text\n\nNakaso K, Horikoshi Y, Takahashi T, et al.: Estrogen receptor-mediated effect of ÎŽ-tocotrienol prevents neurotoxicity and motor deficit in the MPTP mouse model of Parkinsonâs disease. Neurosci. Lett. 2016; 610: 117â122. PubMed Abstract | Publisher Full Text\n\nTocotrienols in parkinson's disease (PD) - full text view. Full Text View. http http\n\nSen CK, Khanna S, Roy S, et al.: Molecular basis of vitamin E action. J. Biol. Chem. 2000; 275(17): 13049â13055. Publisher Full Text\n\nKhanna S, Roy S, Slivka A, et al.: Neuroprotective Properties of the Natural Vitamin E α-Tocotrienol. Stroke. 2005; 36(10): 2258â2264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLa Rosa P, Petrillo S, Turchi R, et al.: The NRF2 induction prevents ferroptosis in Friedreich's ataxia. Redox Biol. 2021; 38: 101791. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZesiewicz T, Salemi JL, Perlman S, et al.: Double-blind, randomized and controlled trial of EPI-743 in Friedreich's ataxia. Neurodegener Dis. Manag. 2018; 2018(8): 233â242.\n\nBolotta A, Pini A, Abruzzo PM, et al.: Effects of tocotrienol supplementation in Friedreichâs ataxia: A model of oxidative stress pathology. Exp. Biol. Med. 2019; 245: 201â212.\n\nShaikh SA, Varatharajan R, Muthuraman A: Palm oil derived tocotrienol-rich fraction attenuates vascular dementia in type 2 diabetic rats. Int. J. Mol. Sci. 2022; 23(21): 13531. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSato Y, Hagiwara K, Arai H, et al.: Purification and characterization of the α-tocopherol transfer protein from rat liver. FEBS Lett. 1991; 288(1-2): 41â45. 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Lipids. 2011; 47(2): 129â139. PubMed Abstract | Publisher Full Text\n\nZiegler M, Wallert M, Lorkowski S, et al.: Cardiovascular and metabolic protection by Vitamin E: A matter of treatment strategy? Antioxidants. 2020; 9(10): 935. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMadkour LH: Biotechnology of Nanostructures Micronutrients Vitamins for Human Health. J. NanoSci. Res. Rep. 2021; 2(5): 358â371. Publisher Full Text\n\nMeganathan P, Jabir R, Fuang H, et al.: A new formulation of Gamma Delta Tocotrienol has superior bioavailability compared to existing Tocotrienol-Rich Fraction in healthy human subjects. Sci. Rep. 2015; 5(1): 13550. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHsu CY, Wang PW, Alalaiwe A, et al.: Use of lipid nanocarriers to improve oral delivery of vitamins. Nutrients. 2019; 11(1): 68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbuasal B, Qosa H, Sylvester P, et al.: Comparison of the intestinal absorption and bioavailability of γ-tocotrienol and α-tocopherol: in vitro, in situ and in vivo studies. Biopharm. Drug Dispos. 2012; 33(5): 246â256. PubMed Abstract | Publisher Full Text\n\nIrÃas-Mata A, Sus N, Flory S, et al.: α-Tocopherol transfer protein does not regulate the cellular uptake and intracellular distribution of α- and γ-tocopherols and -tocotrienols in cultured liver cells. Redox Biol. 2018; 19: 28â36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nManiam G, Mai C, Zulkefeli M, et al.: Challenges and Opportunities of Nanotechnology as Delivery Platform for Tocotrienols in Cancer Therapy. Front. Pharmacol. 2018; 9: 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarcÃa-Pinel B, Porras-Alcalá C, Ortega-RodrÃguez A, et al.: Lipid-Based Nanoparticles: Application and Recent Advances in Cancer Treatment. Nanomaterials. 2019; 9(4): 638. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaez V, Souza ID, Mansur CR: Lipid nanoparticles (SLN & NLC) for delivery of Vitamin E: A comprehensive review. Int. J. Cosmet. Sci. 2018; 40(2): 103â116. Publisher Full Text\n\nAlqahtani S, Simon L, Astete C, et al.: Cellular uptake, antioxidant and antiproliferative activity of entrapped α-tocopherol and γ-tocotrienol in poly (lactic-co-glycolic) acid (PLGA) and chitosan covered PLGA nanoparticles (PLGA-Chi). J. Colloid Interface Sci. 2015; 445: 243â251. PubMed Abstract | Publisher Full Text\n\nMohamad NV, Ima-Nirwana S, Chin KY: Therapeutic potential of annatto tocotrienol with self-emulsifying drug delivery system in a rat model of postmenopausal bone loss. Biomed. Pharmacother. 2021; 137: 111368. PubMed Abstract | Publisher Full Text\n\nYap S, Yuen K, Lim A: Influence of route of administration on the absorption and disposition of α-, γ- and ÎŽ-tocotrienols in rats. J. Pharm. Pharmacol. 2003; 55(1): 53â58. PubMed Abstract | Publisher Full Text\n\nEfendy Goon D, Sheikh Abdul Kadir S, Latip N, et al.: Palm Oil in Lipid-Based Formulations and Drug Delivery Systems. Biomolecules. 2019; 9(2): 64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee Y, Seow E, Lim S, et al.: Formulation and In Vivo Evaluation of a Solid Self-Emulsifying Drug Delivery System Using Oily Liquid Tocotrienols as Model Active Substance. Pharmaceutics. 2021; 13(11): 1777. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlqahtani S, Alayoubi A, Nazzal S, et al.: Enhanced Solubility and Oral Bioavailability of γ-Tocotrienol Using a Self-Emulsifying Drug Delivery System (SEDDS). Lipids. 2014; 49(8): 819â829. PubMed Abstract | Publisher Full Text\n\nAhsan H, Ahad A, Iqbal J, et al.: Pharmacological potential of tocotrienols: a review. Nutr. Metab. 2014; 11(1): 52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMudenur C, Ghosh T, Katiyar V: Nanodelivery system of bioactive compounds in edible food packaging. Nanotechnology in Edible Food Packaging, Materials Horizons: From Nature to Nanomaterials. Singapore: Springer; 2021; pp. 273â298. Publisher Full Text\n\nFernandes F, Dias-Teixeira M, Delerue-Matos C, et al.: Critical review of lipid-based nanoparticles as carriers of neuroprotective drugs and extracts. Nanomaterials. 2021; 11(3): 563. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCuomo F, Iacovino S, Sacco P, et al.: Progress in colloid delivery systems for protection and delivery of phenolic bioactive compounds: Two study casesâHydroxytyrosol and Curcumin. Molecules. 2022; 27(3): 921. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen X, Zou LQ, Niu J, et al.: The stability, sustained release and cellular antioxidant activity of curcumin nanoliposomes. Molecules. 2015; 20(8): 14293â14311. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen MH, Chiang BH: Modification of curcumin-loaded liposome with edible compounds to enhance ability of crossing blood brain barrier. Colloids Surf. A Physicochem. Eng. Asp. 2020; 599: 124862. Publisher Full Text\n\nBonferoni M, Rossi S, Sandri G, et al.: Nanoemulsions for ânose-to-brainâ drug delivery. Pharmaceutics. 2019; 11(2): 84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar A, Ahuja A, Ali J, et al.: Curcumin-loaded lipid nanocarrier for improving bioavailability, stability and cytotoxicity against malignant glioma cells. Drug Deliv. 2014; 23(1): 214â229. PubMed Abstract | Publisher Full Text\n\nRahiminejad A, Dinarvand R, Johari B, et al.: Preparation and investigation of indirubin-loaded SLN nanoparticles and their anti-cancer effects on human glioblastoma U87MG cells. Cell Biol. Int. 2018; 43(1): 2â11. 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Publisher Full Text\n\nMd S, Mustafa G, Baboota S, et al.: Nanoneurotherapeutics approach intended for direct nose to brain delivery. Drug Dev. Ind. Pharm. 2015; 41(12): 1922â1934. PubMed Abstract | Publisher Full Text\n\nKumar S, Dang S, Nigam K, et al.: Selegiline nanoformulation in attenuation of oxidative stress and upregulation of dopamine in the brain for the treatment of parkinson's disease. Rejuvenation Res. 2018; 21(5): 464â476. PubMed Abstract | Publisher Full Text\n\nMadane RG, Mahajan HS: Curcumin-loaded nanostructured lipid carriers (NLCS) for nasal administration: Design, characterization, and in vivo study. Drug Deliv. 2014; 23(4): 1326â1334. PubMed Abstract | Publisher Full Text\n\nDamanhuri H, Abdul Rahim N, Wan Nasri W, et al.: Tocotrienol-rich fraction supplementation modulates antioxidant enzymes activity and reduces DNA damage in APPswe/PS1dE9 Alzheimerâs Disease Mouse Model. 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PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "188254",
"date": "03 Aug 2023",
"name": "Abhijnya Kanugovi",
"expertise": [
"Reviewer Expertise Cell biology",
"Therapeutics",
"Stem cells",
"Neurology",
"Cancer."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article clearly compiles all the relevant literature for using tocotrienol as a drug to target neurodegenerative diseases. The article clearly describes the medicinal values of Tocotrienol, and the problems encountered in drug delivery. While addressing the problem of Tocotrienol delivery, the authors clearly describe the existing methods and their shortcomings and suggest strategies to solve the issue. In summary, the review article is very comprehensive for researchers studying in this field.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
},
{
"id": "204574",
"date": "19 Sep 2023",
"name": "Arunachalam Muthuraman",
"expertise": [
"Reviewer Expertise Neuropharmacology."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall comments: Mathew et al. described in this manuscript: the role of tocotrienol drug delivery pattern for the exploration of anti-inflammatory actions for the management of neurodegenerative diseases. The authors also described the pharmacokinetic profile for the exploration of tocotrienol drug delivery techniques. The major limitation is manuscript has expressed mixed action of T3 on neurodegenerative and non-neurodegenerative disorders. Multiple statements are vague. The overall manuscript needs to improve and it may help in a better understanding, of those working in this field of research.\nSpecific comments:\nMultiple sections like Introduction, anti-inflammatory properties, etc. have too many small paragraphs. Need to be concise with limited paragraphs with a logical sequence of the statements.\n\nFigure 2. The target must be cited as evidence of T3 actions. Need to highlight based on actions on neurodegenerative disease.\n\nFigure 3. The tag created in Biorender.com can be removed; can be mentioned in figure legends.\n\nEach neurodegenerative section end needs to conclude the T3 action on respective neurodegenerative disorders.\n\nTable 2 references can be placed as the last column.\n\nT3 action on VaD is covered limited; needs to expand similarly to other disorders.\n\nThe consequences and challenges of T3 action for neurodegenerative disorders and the importance of Drug delivery systems application for T3 therapy need to be explained before the bioavailability section.\n\nThe statement âOther parenteral vitamin E administration modes include intraperitoneal, intravenous, intramuscular, and subcutaneous injections.3â is incomplete hanging. The various route-based PK profiles of T3 must be discussed.\n\nWhat is the need for Curcumin discussion in the Liposomes and nanoliposomes formulations?\n\nFuture directions and conclusion author emphasized the nano-delivery of intranasal administration T3. These sections must cover all other nano-delivery concepts or the title must be changed based on the intranasal nano-delivery systems.\nMinor comments\nShould be reducing the appearance of too many small paragraphs.\n\nText alignment and typo errors need to be rectified.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": []
},
{
"id": "204573",
"date": "16 Sep 2024",
"name": "Rahul Shukla",
"expertise": [
"Reviewer Expertise Drug Delivery Systems for Neurodegenerative disorders and cancer"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article, \"Exploring the anti-inflammatory activities, mechanism of action and prospective drug delivery systems of tocotrienol to target neurodegenerative diseases\" is well-written. The paper is an important addition to the literature. They can further read various articles on neurodegenerative disorders in recent years relevant to drug delivery. There are a few grammatical mistakes in the manuscript, could you correct these errors for clarity of content? More examples need to be incorporated in the manuscript in various delivery systems. It can be indexed after minor revision.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-338
|
https://f1000research.com/articles/11-1081/v1
|
21 Sep 22
|
{
"type": "Brief Report",
"title": "Improving the predictive power of xenograft and syngeneic anti-tumour studies using mice humanised for pathways of drug metabolism",
"authors": [
"Colin J. Henderson",
"Aileen W. McLaren",
"Yury Kapelyukh",
"C. Roland Wolf",
"Colin J. Henderson",
"Aileen W. McLaren",
"Yury Kapelyukh"
],
"abstract": "Drug development is an expensive and time-consuming process, with only a small fraction of drugs gaining regulatory approval from the often many thousands of candidates identified during target validation. Once a lead compound has been identified and optimised, they are subject to intensive pre-clinical research to determine their pharmacodynamic, pharmacokinetic and toxicological properties, procedures which inevitably involve significant numbers of animals - mainly mice and rats, but also dogs and monkeys in much smaller numbers and for specific types of drug candidates. Many compounds that emerge from this process, having been shown to be safe and efficacious in pre-clinical studies, subsequently fail to replicate this outcome in clinical trials, therefore wasting time, money and, most importantly, animals.\nThe poor predictive power of animal models in pre-clinical studies is predominantly due to lack of efficacy or safety reasons, which in turn can be attributed mainly to the significant species differences in drug metabolism between humans and animals. To circumvent this, we have developed a complex transgenic mouse model â 8HUM - which faithfully replicates human Phase I drug metabolism (and its regulation), and which will generate more human-relevant data [REFINEMENT] from fewer animals [REDUCTION] in a pre-clinical setting and reduce attrition in the clinic.\nOne key area for the pre-clinical application of animals in an oncology setting â almost exclusively mice - is their use in anti-tumour studies. We now further demonstrate the utility of the 8HUM mouse using a murine melanoma cell line as a syngeneic tumour and also present an immunodeficient version 8HUM_Rag2-/- - for use in xenograft studies. These models will be of significant benefit not only to Pharma for pre-clinical drug development work, but also throughout the drug efficacy, toxicology, pharmacology, and drug metabolism communities, where fewer animals will be needed to generate more human-relevant data.",
"keywords": [
"humanised P450 model",
"transgenic mice",
"drug metabolism",
"drug development",
"xenograft",
"chemotherapy",
"combination chemotherapy"
],
"content": "Abbreviations\n\nPDX: patient-derived xenograft\n\np.o.: per os, oral gavage\n\ns.c.: sub-cutaneous\n\n\nIntroduction\n\nThe pre-clinical stage of drug development provides crucial information for the decision process as to whether a drug candidate will proceed to âfirst in humanâ and phased clinical trials.1 The failure rate through the pre-clinical stages of drug development can be high and many candidate molecules taken forward to clinical trials subsequently fail to recapitulate the safety profile and efficacy found in animal studies.2â5 There are many reasons for this, but significant species differences in drug metabolism between animals (rats, mice) and humans â with concomitant changes in pharmacokinetics, metabolite profiles, toxicokinetics and pharmacodynamics â are key components underlying the observed failure rates.5 We have developed a sophisticated transgenic mouse model in which the major human drug metabolising enzymes â and the transcription factors regulating their expression â replace their mouse counterparts. In a previous report on this humanised mouse model6 we show, using model compounds and anti-cancer drugs, that drug metabolism and disposition in the 8HUM mouse more closely reflects that found in humans. Given the growing importance of drug combinations in cancer therapy,7 it is clear that a genetically engineered mouse model such as 8HUM could play a pivotal role in the development of such combinations.8\n\nWhile some pre-clinical work is carried out in vitro, using a variety of cell lines including immortalised human cells, much â and arguably the most important â is carried out in animals, mainly rodents but also dogs, and for certain types of candidate molecules, primates. One such in vivo use in the pre-clinical setting is syngeneic or xenograft work, where anti-tumour efficacy of drug candidates is tested alone or in drug combinations. In a syngeneic model, murine cell lines are implanted subcutaneously or orthotopically and tumour response to candidate drugs tested. Whilst such experiments can account for the effect of immune system, the genetic background of the tumour cells - which must match that of the host animal used â can also give rise to disparate results. More recently xenograft models have come to the fore, where immunodeficient mouse lines are able to grow human tumours either from existing immortalised cell lines or via fresh tissue as patient-derived xenografts (PDX).9,10 Despite lack of a competent immune system and any issues that this may potentially cause in the interpretation of results, the latter are growing in use, a good example being the EurOPDX consortium, who have a database of PDX models to share with the research community.11 Notwithstanding extensive xenograft use in various guises, such models still have issues arising from retention of murine drug metabolism and disposition.8\n\nIn this brief report we showcase a modification of the humanised 8HUM model in which we have generated a compromised immune system by deleting the Rag2 locus. Using murine and human melanoma cell lines in 8HUM_Rag2-/- mice, we show tumour growth in a syngeneic and xenograft setting, respectively, and demonstrate in vivo sensitivity to dabrafenib and trametinib, drugs currently used in combination as standard of care in the treatment of metastatic melanoma. Together, these models have the potential to significantly reduce the number of animals used in the pre-clinical stages of drug development, while generating more human-relevant data and thus improving the chances of a candidate drug replicating a positive pre-clinical finding in successful clinical trials.\n\n\nMethods\n\nUnless specifically stated, reagents used in these studies were purchased from Sigma-Aldrich (Dorset, UK).\n\nTransgenic mice â 8HUM - extensively humanized for the major cytochrome P450 enzymes in Phase I drug metabolism, along with the transcription factors regulating their expression, have previously been described6 and were generated in a collaboration between CXR Biosciences and Taconic Biosciences funded through the Scottish Government ITI, with CRW as one of the principal investigators.\n\nThirty-five murine genes (the Cyp2c (except Cyp2c44), Cyp2d and Cyp3a murine gene clusters and transcription factors Car and Pxr) were replaced by eight human genes (CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP3A4, CYP3A7, CAR, PXR). Expression of human P450 genes was from the human promotor, except for CYP2C9, which was driven by the albumin promotor, and CYP1A1, CYP1A2, CAR and PXR which were driven off the corresponding murine promoters.\n\n8HUM mice were further genetically altered by deleting Rag2 using CRISPR/cas9-mediated gene editing in 8HUM zygotes (Taconic Biosciences GmbH, Germany). Breeding of mice from this process was carried out to re-generate 8HUM mice with a homozygous deletion of Rag2-8HUM_Rag2-/--rendering the line immunodeficient for xenograft studies.\n\nAnimals were on a C57BL/NTac background and were bred, and experimental work carried out in the Medical School Resource Unit, University of Dundee. Mice were held at positive pressure in Techniplast Sealsave BlueLine micro-isolator cages, with Eco-Pure chip7D bedding (Datesand Group, UK) and ad libitum access to water and food-RM1 for maintenance, RM3 for breeding (Special Diet Services, UK). Temperature (20â24oC) and relative humidity (45â65%) were maintained in a 12-hour light-dark environment.\n\nAll animal work was approved by the Welfare and Ethical Treatment of Animals Committee, under Home Office Project (PAFCCC160) and personal licences (I94242D3D, IDFA32717, I372C0F97) under the Animals (Scientific Procedures) Act 1986, as amended by EU Directive 2010/63/EU.\n\nEvery effort was made to ameliorate animal suffering. Animals were inspected regularly by trained and experienced staff, with 24-hour access to veterinary advice, and consideration was given to socialisation by allowing time for settling into new (experimental) groups. In addition, environmental enrichment was routinely added to cages in the form of red plastic tunnels or nests, chew sticks and diet was supplemented by sunflower seeds.\n\nOn study completion, animals were sacrificed by exposure to a rising concentration of CO2 and death confirmed by exsanguination, according to Schedule 1 of the Animals (Scientific Procedures) Act 1986.\n\nAdult female (>8 <22 weeks) mice were randomly allocated into control or experimental groups and allowed to adapt to their social setting for 7 d before study start. Cages were adjacent to each other on the same level of a ventilated rack, in the same room, for the study duration.\n\nNeither animal staff nor experimenters were blinded to the identity of the mice or the experimental group in which they were placed, before, during or after the study.\n\nSample size: Although this work was considered preliminary in nature and the studies carried out as pilots, group sizes of n = 3â5 were used, following consideration of power calculations using G*Power,12 with an effect size of 1.75 and power of 80%.\n\nData analysis: Dependencies of calculated tumour volumes versus time were analysed by non-linear regression using exponential growth and exponential decay functions (GraphPad Prism v6.05 software, Graphpad, US). Alternative, open source, software â R â can be found at https://www.r-project.org. Rate constants in both functions were constrained to positive values to maintain consistency with the function name. Values for plateau parameters in exponential decay function were set to zero.\n\nA375 human melanoma cells (ATCC: CRL-1619; RRID:CVCL_UD29) and 5555 murine melanoma cells13â15 were grown as directed, with the latter subject to commercial murine pathogen testing (IDEXX Bioanalytics GmbH, Germany) and both to in-house mycoplasma testing (MycoAlert Mycoplasma Detection kit, Lonza Rockland, USA) before use. Passage number was recorded for each study.\n\nCell lines were harvested on the morning of the study, kept on ice and transferred to the animal facility for use within 1 h. All animal work was carried out in the sterile environment of a Tecniplast CS5 Evo Changing station.\n\nMice were weighed, fur removed on one/both flanks by electric shaver and placed individually in a red plastic inhalation chamber connected to an anaesthetic machine (Vet-Tech Solutions, Congleton, UK), to which was connected an anaesthetic maintenance tube running into the changing station. General anaesthesia was induced using an isoflurane (Piramal Critical Care, UK)/oxygen mixture in a Series 3 vapouriser (O2 flow rate 2l/min, isoflurane 3.5â4%) and maintained when the mouse was removed from the chamber by lying the animal on its front, snout placed just inside the end of the anaesthetic maintenance tube and the isoflurane/oxygen flow switched to the tube (O2 flow rate 1.5l/min, isoflurane 1.5â2.5%). Prepared cells (3.5â5 à 106, 100 ÎŒl in DMEM (Thermofisher Scientific, UK) were taken up in a 1ml plastic syringe and injected subcutaneously (s.c.) to one or both flanks using a 25mm/23G needle [Optional: cells can be re-suspended in ECM (Sigma), diluted 1:1 with DMEM]. This procedure routinely took <3 min. Immediately after injection, the mouse was returned to its home cage, placed on its front and monitored during recovery, which routinely took <5 min. [Optional: s.c. injection may be carried out immediately after removing mouse from the inhalation chamber, while still under general anaesthesia. However, particularly with immortalised human tumours, care must be taken to avoid self-injection; on safety grounds the maintenance anaesthesia route is strongly recommended.]\n\nIn addition to routine welfare monitoring, mice were weighed and checked for tumour growth daily. Body weight was used in conjunction with a body scoring system.16 Deviation from normal health, >10% body weight loss, or a body condition score of 2 or less was referred to the University Vet or Welfare Officer. If any animal appeared distressed, or a tumour ulcerated, the animal was removed from study and killed by a Schedule 1 method.\n\nOnce established, tumours were measured twice in two dimensions (maximum breadth and length) using digital calipers, by the same person to avoid interindividual variation. Treatment was also started at this point, mice receiving either vehicle or drug daily (p.o.).\n\nDabrafenib methanesulfonate (LC Laboratories, MA, USA) was prepared as a 6.3 mg/ml suspension in vehicle (0.5%(w/v) hydroxypropylmethylcellulose, 0.2%(v/v) Tween-80) after 10 min sonication in a water bath and administered daily (p.o.) at 5 ml/kg, and a dose of 31.5 mg/kg. This is equivalent to approximately 150 mg of dabrafenib base for a 70 kg human,17 approximately half of the recommended daily dose (https://dailymed.nlm.nih.gov/).\n\nTrametinib (LC Laboratories, MA, USA) was prepared as a 0.07048 mg/ml suspension in vehicle (0.5%(w/v) hydroxypropylmethylcellulose, 0.5%(v/v) Tween-20) and administered daily (p.o.) at 5 ml/kg, and a dose of 0.3524 mg/kg. This is equivalent to 2 mg of trametinib for a 70 kg human,17 which is the recommended daily dose (https://dailymed.nlm.nih.gov/).\n\nTumour volume was estimated using the formula ((width*width)*length)/2.18 Tumour length was also monitored, and mice in which tumour length reached 15mm (either individually or in total if tumours on both flanks) were sacrificed by a Schedule 1 method and blood, tissues and tumours harvested as appropriate for downstream analysis.\n\n\nResults\n\n8HUM mice were used to determine syngeneic growth of the murine melanoma cell line, 5555, derived from a C57BL/6_BRAF+/LSL-BRAFV600E;Tyr::CreERT2+/o transgenic model13 and reported by Hirata et al. as being sensitive to the selective BRAF inhibitor, and vemurafenib precursor, PLX472019 in vitro, but refractory to this drug in vivo.14 More recently, the second-generation mutant BRAF inhibitor dabrafenib (in combination with the MEK inhibitor trametinib) has become standard of care in UK and Europe in the treatment of unresectable or metastatic BRAFV600 mutant melanoma (https://www.nice.org.uk/guidance/).20 All data for this report are available online at.21,22 As shown in Figure 1, 5555 cells were injected s.c. into one flank of adult female 8HUM mice, divided into two groups of five mice. After five days tumours had established in each animal and were measurable, at which point daily oral treatment with either vehicle or dabrafenib was started. While tumours in vehicle-treated mice continued to grow over the following two weeks, over the same period tumours in mice treated with dabrafenib became almost undetectable. These data demonstrate that C57BL/6-origin tumours can be grown in a syngeneic manner, and that the BRAFV600 mutant murine melanoma cell line is exquisitely sensitive to the BRAF inhibitor dabrafenib.\n\nAdult female 8HUM_Rag2-/- mice (18â21 w, n = 5) were injected s.c. in one flank with 3.5 à 106 5555 murine melanoma cells, in 100 ÎŒl ECM diluted 1:1 with DMEM. Tumours were allowed to establish and on day five after implantation daily treatment was commenced with either vehicle (0.5% (w/v) hydroxypropylmethylcellulose, 0.2% (v/v) Tween 80; closed circles) or dabrafenib methanesulfonate (in vehicle, open circles) suspended at 6.3 mg/ml and administered at 5 ml/kg, such that dabrafenib dose administered was 31.5 mg/kg (arrow). Tumour measurements were taken three times weekly, then daily as required, and tumour volume calculated as detailed in Methods section. The study was terminated 15 days after implantation of cells.\n\nData shown are mean tumour volume ± SEM.\n\nBy creating an immunodeficient variant of the 8HUM mouse line, where the Rag2 locus is deleted, we extended work to xenografts with the BRAF mutant human melanoma cell line, A375. Figure 2 shows change in total mean tumour volume following s.c injection of A375 cells injected into both flanks of adult female 8HUM_Rag2-/- mice. Daily treatment of these mice started 28 d after injection of cells, either with vehicle or dabrafenib (arrow); while the tumours in the former group continued to grow, tumours in the mice treated with the BRAF inhibitor shrank in size until by d35 (at which point the vehicle-treated mice had to be sacrificed due to tumour size) there was a significant difference in the treatment effect between the two groups (Figure 2). The data from vehicle group follows the exponential growth dependency and does not fit with the exponential decay function. Conversely, the dabrafenib group data follows exponential decay dependency and does not fit the exponential growth function. These data clearly demonstrate not only that it is possible to grow a human melanoma cell line as a xenograft in this immunodeficient version of the 8HUM mouse model, but it is also possible to demonstrate sensitivity of A375 tumours to BRAF inhibitors.\n\nAdult female 8HUM mice (11â19 w, n = 3) were injected s.c. in both flanks with 4.4 à 106 A375 melanoma cells, in 100 ÎŒl DMEM. Tumours were allowed to establish and on day 28 after implantation daily treatment was commenced with either vehicle (0.5% (w/v) hydroxypropylmethylcellulose, 0.2% (v/v) Tween-80; closed circles) or dabrafenib (in vehicle, open circles) suspended at 6.3 mg/ml and administered at 5 ml/kg, such that dabrafenib dose administered was 31.5 mg/kg (arrow). Tumour measurements were taken three times weekly, then daily as required, and total volume of tumours on both flanks was calculated as detailed in Methods section. The study was terminated on d 35 after implantation of cells, although one vehicle-treated animal had to be removed from the study on d 29 as its total tumour size approached the maximum permitted under legislation.\n\nData shown are mean tumour volume ± SEM.\n\nDabrafenib is used in a clinical setting in combination with the MEK inhibitor trametinib. We tested the ability of trametinib to stop tumour growth, using A375 cells injected s.c. in the flank of adult female 8HUM_Rag2-/- mice (Figure 3). Daily treatment with vehicle or drug commenced on d 22 after cell injection (arrow), and in the following period tumours in mice treated with vehicle continued to grow until by d 30 they had reached the maximum size permitted. At this point (Figure 3, STOP) tumours in the 8HUM_Rag2-/- mice had regressed to the point where they were essentially undetectable, and trametinib treatment was stopped. Interestingly, over the following 10 days, tumours began to regrow in the absence of treatment until they were again palpable and measurable, and continued to grow over the next week or so until the study was terminated on d 48 (Figure 3).\n\nAdult female 8HUM mice (8â18w, n = 3 or 4) were injected s.c. in one flank with 5 à 106 A375 melanoma cells, in 100 ÎŒl DMEM. Tumours were allowed to establish and on day 22 after implantation daily treatment was commenced with either vehicle (0.5% (w/v) hydroxypropylmethylcellulose, 0.2% (v/v) Tween 80; closed circles) or trametinib (in vehicle, open circles) suspended at 0.07 mg/ml and administered at 5 ml/kg, such that trametinib dose administered was 0.35 mg/kg (arrow). Tumour measurements were taken three times weekly, then daily as required, and tumour volume calculated as detailed in Methods section. Vehicle-treated mice were sacrificed on d 30 after implantation of cells; trametinib treatment was discontinued (STOP) at that time for the drug-treated group to determine whether tumour re-growth would occur in the absence of drug.\n\nData shown are mean tumour volume ± SEM.\n\n\nDiscussion and conclusions\n\nIn a previous publication6 we have demonstrated the utility of the 8HUM model in better predicting human drug metabolism and disposition, and in the current report show how the 8HUM mouse and its immunodeficient variant 8HUM_Rag2-/- are capable of hosting both syngeneic tumours and xenografts, respectively.\n\nWhile numbers of animals used in syngeneic or xenograft work in pre-clinical drug development are difficult to assess, the total will be significant given the number of drug candidates being tested across Pharma at any given time, and such growth of tumours in vivo is also carried out in other research areas, for example, toxicology. A PubMed search for papers published in 2019 found ~5,000 papers containing âxenograftâ in the title or abstract, illustrating the extent to which the 8HUM and 8HUM_Rag2-/- models â modified as appropriate by gene editing to recapitulate disease models - may be able to address 3Rs issues by both refinement: generation of better, more human-relevant data, and reduction â use of fewer animals without loss of statistical power. Pre-clinical use of humanised models to prevent failure of a drug candidate during clinical testing because of species differences in drug disposition would undoubtedly save significant numbers of mice. They will also allow complex drug combinations to be tested and treatment regimens optimised in a manner which is not feasible by clinical trial and reduce the chances of drug-drug interactions.\n\nThe 8HUM has some limitations, and it should be noted that a minor complement of murine P450 enzymes remain, and that the Phase II enzymes are murine. These may potentially contribute to drug disposition, as may other pathways e.g., drug transporters. However, the advent of CRISPR/Cas9, as used here to delete the Rag2 locus in the 8HUM mouse, means that it should be relatively simple to additionally modify the 8HUM model to further enhance versatility.\n\n\nAuthor contributions\n\nConceptualizationââââWolf, Henderson, Kapelyukh\n\nData CurationââââââMcLaren, Henderson, Kapelyukh\n\nFormal analysisâââââKapelyukh, Henderson\n\nFunding acquisitionââââWolf, Henderson\n\nInvestigationââââââKapelyukh, McLaren, Henderson\n\nMethodologyââââââWolf, Kapelyukh, McLaren, Henderson\n\nProject AdministrationââMcLaren, Henderson\n\nResourcesâââââââMcLaren, Henderson\n\nSupervisionââââââWolf, Henderson\n\n\nData availability\n\nFigshare: Underlying data for âImproving the predictive power of xenograft and syngeneic anti-tumour studies using mice humanised for pathways of drug metabolismâ. https://doi.org/10.6084/m9.figshare.20060465.v1.21\n\nThis project contains the following underlying data:\n\n⢠Data file 1: FIG 1 BRI995 Weights&Tumours 280219.xlsx\n\n⢠Data file 2: FIG 2 BRI1102 Weights&Tumour 010421.xlsx\n\n⢠Data file 3: FIG 3 BRI1119 Weights&Tumour 210621.xlsx\n\nFigshare: ARRIVE checklist for âImproving the predictive power of xenograft and syngeneic anti-tumour studies using mice humanised for pathways of drug metabolismâ. https://doi.org/10.6084/m9.figshare.20060465.v1.22\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)",
"appendix": "Acknowledgements\n\nThe staff of the Medical School Resource Unit at the University of Dundee are thanked for their assistance with animal work.\n\nThe mouse lines described in this manuscript were developed under a material transfer agreement between Taconic Biosciences and the University of Dundee.\n\nMurine 5555 cells were a generous gift from Professor Richard Marais, Cancer Research UK Centre, University of Manchester, UK.\n\n\nReferences\n\nHonek J: Preclinical research in drug development. Medical Writing. 2017; 26: 5â8.\n\nDowden H, Munro J: Trends in clinical success rates and therapeutic focus. Nat. Rev. Drug Discov. 2019; 18(7): 495â496. PubMed Abstract | Publisher Full Text\n\nHarrison RK: Phase II and phase III failures: 2013-2015. Nat. Rev. Drug Discov. 2016; 15(12): 817â818. PubMed Abstract | Publisher Full Text\n\nLong JE, Jankovic M, Maddalo D: Drug discovery oncology in a mouse: concepts, models and limitations. Future Sci. OA. 2021; 7(8): FSO737. PubMed Abstract | Publisher Full Text\n\nSun D, Gao W, Hu H, et al.: Why 90% of clinical drug development fails and how to improve it? Acta Pharm. Sin. B. 2022; 12: 3049â3062. PubMed Abstract | Publisher Full Text\n\nHenderson CJ, Kapelyukh Y, Scheer N, et al.: An Extensively Humanized Mouse Model to Predict Pathways of Drug Disposition and Drug/Drug Interactions, and to Facilitate Design of Clinical Trials. Drug Metab. Dispos. 2019; 47(6): 601â615. PubMed Abstract | Publisher Full Text\n\nDuarte D, Vale N: Evaluation of synergism in drug combinations and reference models for future orientations in oncology. Curr. Res. Pharmacol. Drug Discov. 2022; 3: 100110. PubMed Abstract | Publisher Full Text\n\nKersten K, de Visser KE , van Miltenburg MH , et al.: Genetically engineered mouse models in oncology research and cancer medicine. EMBO Mol. Med. 2017; 9(2): 137â153. PubMed Abstract | Publisher Full Text\n\nByrne AT, Alferez DG, Amant F, et al.: Interrogating open issues in cancer precision medicine with patient-derived xenografts. Nat. Rev. Cancer. 2017; 17(4): 254â268. PubMed Abstract | Publisher Full Text\n\nGoto T: Patient-Derived Tumor Xenograft Models: Toward the Establishment of Precision Cancer Medicine. J Pers Med. 2020; 10(3). PubMed Abstract | Publisher Full Text\n\nDudova Z, Conte N, Mason J, et al.: The EurOPDX Data Portal: an open platform for patient-derived cancer xenograft data sharing and visualization. BMC Genomics. 2022; 23(1): 156. PubMed Abstract | Publisher Full Text\n\nFaul F, Erdfelder E, Buchner A, et al.: Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav. Res. Methods. 2009; 41(4): 1149â1160. PubMed Abstract | Publisher Full Text\n\nDhomen N, Reis-Filho JS, da Rocha DS , et al.: Oncogenic Braf induces melanocyte senescence and melanoma in mice. Cancer Cell. 2009; 15(4): 294â303. PubMed Abstract | Publisher Full Text\n\nHirata E, Girotti MR, Viros A, et al.: Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin beta1/FAK signaling. Cancer Cell. 2015; 27(4): 574â588. PubMed Abstract | Publisher Full Text\n\nRoulstone V, Pedersen M, Kyula J, et al.: BRAF- and MEK-Targeted Small Molecule Inhibitors Exert Enhanced Antimelanoma Effects in Combination With Oncolytic Reovirus Through ER Stress. Mol. Ther. 2015; 23(5): 931â942. Publisher Full Text\n\nUllman-Cullere MH, Foltz CJ: Body condition scoring: a rapid and accurate method for assessing health status in mice. Lab. Anim. Sci. 1999; 49(3): 319â323. PubMed Abstract\n\nReagan-Shaw S, Nihal M, Ahmad N: Dose translation from animal to human studies revisited. FASEB J. 2008; 22(3): 659â661. PubMed Abstract | Publisher Full Text\n\nFaustino-Rocha A, Oliveira PA, Pinho-Oliveira J, et al.: Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Anim (NY). 2013; 42(6): 217â224. PubMed Abstract | Publisher Full Text\n\nTsai J, Lee JT, Wang W, et al.: Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl. Acad. Sci. U. S. A. 2008; 105(8): 3041â3046. PubMed Abstract | Publisher Full Text\n\nLong GV, Stroyakovskiy D, Gogas H, et al.: Dabrafenib and trametinib versus dabrafenib and placebo for Val600 BRAF-mutant melanoma: a multicentre, double-blind, phase 3 randomised controlled trial. Lancet. 2015; 386(9992): 444â451. PubMed Abstract | Publisher Full Text\n\nHenderson C, Wolf CR, Kapelyukh Y, et al.: FIGS 1-3 NC3Rs F1000 submission 122987.xlsx. figshare. [Dataset].2022. Publisher Full Text\n\nHenderson C, Wolf CR, Kapelyukh Y, McLaren A, et al.: ARRIVE Checklist - Full.pdf. figshare. [Dataset].2022. Publisher Full Text"
}
|
[
{
"id": "159796",
"date": "19 Jan 2023",
"name": "Russ Prough",
"expertise": [
"Reviewer Expertise drug metabolism",
"CYPs"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript in general is clearly written and leads the reader through the results. The statistical details providing valid conclusions is well documented. The Authors have developed a unique murine model to provided data on human metabolism. Past reports document the utility of model as being useful to document. A couple specific questions arise:\n1. If the authors had used the original mouse line (with the Rag2 gene present), would the results have been the same or less sensitive to the anti-cancer drugs?\n\n2.  If that data exists, has it been published? If not would inclusion of such data strengthen the current manuscript. This would document the importance of the Rag2 KO.\n3. Some anti-cancer drugs induced their own metabolism. Has this been studied for Dabrafenib or Tremetinib?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9425",
"date": "27 Mar 2023",
"name": "Colin Henderson",
"role": "Author Response",
"response": "Point 1: If the authors had used the original mouse line (with the Rag2 gene present), would the results have been the same or less sensitive to the anti-cancer drugs? The human cell line (A375) would not have grown in an immunocompetent 8HUM line. Point 2:  If that data exists, has it been published? If not would inclusion of such data strengthen the current manuscript. This would document the importance of the Rag2 KO. Data does not exist for the reason outlined above. Point 3: Some anti-cancer drugs induced their own metabolism. Has this been studied for Dabrafenib or Trametinib? Dabrafenib is principally metabolised by CYP2C8 and CYP3A4, and in common with many drugs, can induce its own metabolism (see - Clinical Pharmacokinetics and Pharmacodynamics of Dabrafenib, (2019) Puszkiel, A. et al., PMID: 30094711). With regard to Trametinib, the position is less clear. We have also been able to reproduce the known induction of P450 CYP3A4 by dabrafenib in the humanized mice, further demonstrating the utility of the model in anticancer drug development (unpublished data), and a sentence to this effect has been added to the text of the Results section."
}
]
},
{
"id": "160109",
"date": "13 Feb 2023",
"name": "Chris Goldring",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important preliminary report that will be of value to the academic and pharmaceutical drug development scientists.\n\nSome specific points: Methodologically, this looks to be a preliminary but robust report. The lack of characterisation of the tumours post-mortem is an issue that the authors could address using conventional histological characterisation. This is not a request that should not significantly impede time to publication, but would add more depth to the findings.\nIn the abstract ââThe poor predictive power of animal models in pre-clinical studies is predominantly due to lack of efficacy or safety reasons, which in turn can be attributed mainly to the significant species differences in drug metabolism between humans and animals.â This is largely correct for older traditional investigative compounds, however as drug development pipelines have evolved, especially with the advent of more large molecule development, the issue of target-drug conservation of pharmacodynamic effect becomes very relevant. The authors should make some account of this (given that the two agents looked at in this report are conventional small molecule inhibitors).\nPharma largely uses rat models, for various reasons. The authors should note this in the context of their own mouse model.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9426",
"date": "27 Mar 2023",
"name": "Colin Henderson",
"role": "Author Response",
"response": "Point 1: Pathological characterisation It should be noted that the xenografts used in this study were derived from a human cell line and not from a solid tumour. In addition, tumour samples are not available for the drug treated groups because of the effectiveness of the treatments used. On this basis we feel adding histopathological data would add little to the main conclusions of the study. Point 2: Species differences in drug metabolism remain a significant impediment in drug development In the light of the Reviewerâs comment, we have changed the sentence to read \"Species differences in drug metabolism can be a major impediment to drug discovery programmes, for example in the demonstration of drug efficacy as a consequence of high rates of metabolism in rodents. Furthermore, the spectrum of metabolites produced, which can be pharmacologically active, is often significantly different between humans and rodents. These factors can confound the extrapolation of animal data to man particularly when drug combination therapies are being testedâ. Point 3: The Reviewer is correct to point out that both rats and mice are used by industry in the drug development process. However, the utility of mice for growing xenografts, including patient-derived xenografts, and the development of âmouse clinical trialsâ, along with the more advanced use of mice in genetically engineered models (such as 8HUM) means that increasingly mice are being considered more useful for drug development, especially in oncology. A sentence to this effect has been placed in the Discussion, and additional references added."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1081
|
https://f1000research.com/articles/11-1274/v1
|
08 Nov 22
|
{
"type": "Research Article",
"title": "Targeting Acanthamoeba proteins interaction with flavonoids of Propolis extract by in vitro and in silico studies for promising therapeutic effects",
"authors": [
"Imran Sama-ae",
"Suthinee Sangkanu",
"Abolghasem Siyadatpanah",
"Roghayeh Norouzi",
"Julalak Chuprom",
"Watcharapong Mitsuwan",
"Sirirat Surinkaew",
"Rachasak Boonhok",
"Alok K. Paul",
"Tooba Mahboob",
"Najme Sadat Abtahi",
"Tajudeen O. Jimoh",
"Sónia M.R. Oliveira",
"Madhu Gupta",
"Chea Sin",
"Maria de Lourdes Pereira",
"Polrat Wilairatana",
"Christophe Wiart",
"Mohammed Rahmatullah",
"Karma G. Dolma",
"Veeranoot Nissapatorn",
"Imran Sama-ae",
"Suthinee Sangkanu",
"Abolghasem Siyadatpanah",
"Roghayeh Norouzi",
"Julalak Chuprom",
"Watcharapong Mitsuwan",
"Sirirat Surinkaew",
"Rachasak Boonhok",
"Alok K. Paul",
"Tooba Mahboob",
"Najme Sadat Abtahi",
"Tajudeen O. Jimoh",
"Sónia M.R. Oliveira",
"Madhu Gupta",
"Chea Sin",
"Maria de Lourdes Pereira",
"Polrat Wilairatana",
"Christophe Wiart",
"Mohammed Rahmatullah",
"Karma G. Dolma"
],
"abstract": "Background: Propolis is a natural resinous mixture produced by bees. It provides beneficial effects on human health in the treatment/management of many diseases. The present study was performed to demonstrate the anti-Acanthamoeba activity of ethanolic extracts of Propolis samples from Iran. The interactions of the compounds and essential proteins of Acanthamoeba were also visualized through docking simulation. Methods: The minimal inhibitory concentrations (MICs) of Propolis extract against Acanthamoeba trophozoites and cysts was determined in vitro. In addition, two-fold dilutions of each of the agents were tested for encystment, excystment and adhesion inhibitions. Three major compounds of Propolis extract such as chrysin, tectochrysin and pinocembrin have been selected in molecular docking approach to predict the compounds that might be responsible for encystment, excystment and adhesion inhibitions of A. castellanii. Furthermore, to confirm the docking results, molecular dynamics (MD) simulations were also carried out for the most promising two ligand-pocket complexes from docking studies. Results: The minimal inhibitory concentrations (MICs) 62.5 and 125 µg/mL of the most active Propolis extract were assessed in trophozoites stage of Acanthamoeba castellanii ATCC30010 and ATCC50739, respectively. At concentrations lower than their MICs values (1/16 MIC), Propolis extract revealed inhibition of encystation. However, at 1/2 MIC, it showed a potential inhibition of excystation and anti-adhesion. The molecular docking and dynamic simulation revealed the potential capability of Pinocembrin to form hydrogen bonds with A. castellanii Sir2 family protein (AcSir2), an encystation protein of high relevance for this process in Acanthamoeba. Conclusions: The results obtained provided a candidate for the development of therapeutic drugs against Acanthamoeba infection. In vivo experiments and clinical trials are necessary to support this claim.",
"keywords": [
"Propolis extract",
"anti-Acanthamoeba activity",
"encystation",
"pinocembrin",
"molecular docking",
"and dynamic simulation"
],
"content": "Introduction\n\nAcanthamoeba, a free-living ameba, is a causative agent of fatal granulomatous amoebic encephalitis (GAE), Acanthamoeba keratitis (AK), Acanthamoeba pneumonia (AP), cutaneous acanthamoebiasis, and disseminated acanthamoebiasis found in humans1. In healthy individuals with contact lenses, Acanthamoeba keratitis is increasingly being recognized as a serious sight-threatening ocular infection in public health worldwide2. Acanthamoeba life cycle includes an active trophozoite stage and a dormant cyst stage. The trophozoite stage is the motile form that acquires nutrients, neutral pH, adequate food supply, ambient temperature, and balanced osmolality, while the cyst is triggered by extreme conditions, such as food crisis, hyper- or hypo-osmolarity, temperature, and excessive acid to basic conditions. Regarding the Acanthamoeba keratitis, the cyst form can be found in the acceptor cornea and is difficult to treat due to the resilient nature of the cyst. Current treatment regimens usually include standard anti-Acanthamoeba drugs, biguanide and diamidine, for an effective treatment against cysts3. However, long-term treatment has also been suggested to induce a resistant Acanthamoeba cyst form due to a non-specific symptom at the early stage of AK, which share other common features, such as eye pain and redness4.\n\nPropolis or bee glue is a mixture of honeybees and natural products of different parts of plants5 that is used for the construction and repairing beehives. Propolis hardens the cell wall of beehives, contributes to an aseptic internal environment6, and acts as a protective barrier against predators. In addition, Propolis property contains several biological activities such as anti-inflammation, anti-proliferation, antioxidant, anti-diabetic activity, and antimicrobial activities7â9.\n\nTherefore, this study sought to evaluate an amoebicidal activity and anti-Acanthamoeba encystation, excystation and anti-adhesion by Propolis extract that could offer an alternative treatment strategy for Acanthamoeba infection. Molecular docking simulation was included to predict a predominant binding mode of small molecules derived from Propolis with essential proteins from Acanthamoeba spp., to identify a relevant stable protein-ligand complex for future drug development.\n\n\nMethods\n\nThree Propolis samples were collected from Sardasht county, Boroujen city and Kermanshah city from Iran. The raw materials of Propolis were cut into small pieces, homogenizing (20 g) with 50 mL absolute ethanol and incubated at room temperature for seven days without shaking. Then, the extract was filtered through Whatman No. 1 filter paper, and the alcoholic extract was evaporated under vacuum with a rotary evaporator until it was dry. Dried extracts were preserved at 4°C and re-suspended in dimethyl sulfoxide (DMSO) at 100 mg/mL concentration before use.\n\nAcanthamoeba castellanii non-pathogenic strain (ATCC 30010) and Acanthamoeba castellanii pathogenic strain (ATCC 50739) were obtained from the American Type Culture Collection (ATCC). Trophozoites were grown in 75 cm2 tissue culture flasks in Peptone Yeast Extract Glucose Broth (PYG) medium containing proteose peptone 0.75% (w/v), yeast extract 0.75% (w/v) and glucose 1.5% (w/v) (purchased from HiMedia Laboratories Pvt.Ltd., Mumbai, India), without shaking at 28°C as described previously10. For cysts, trophozoites were transferred from the PYG medium to the Neffâs encystment medium (NEM) containing 0.1 M KCl, 8 mM MgSO4·7H2O, 0.4 mM CaCl2·2H2O, 1 mM NaHCO3, 20 mM ammediol (purchased from RCI Labscan Limited, Bangkok, Thailand) and were cultured in this medium for seven days to obtain mature cysts. After that, mature cysts were harvested and washed twice using 10 mL sterile phosphate-buffer saline (PBS).\n\nThe MIC was determined by the micro-dilution method using serially diluted (two-fold) Propolis extracts. Determination of the MIC of the Propolis extract was examined according to a previous study10. Stock solution of extracts (4 ÎŒL) were transferred into the first well of 96-well microplates, including 196 ÎŒL PYG medium to obtain a final concentration of 2,000 µg/mL. A two-fold serial dilution of the extracts were prepared in 96-well assay microplates to obtain concentrations in the range of 7.8â1,000 ÎŒg/mL in PYG medium. Then, 100 µL trophozoites or cysts (2Ã105 cells/mL) were added. The final volume in each well was 200 ÎŒL. Plates were incubated for 24 hours at 28°C. The percentage of cell viability was determined using 0.2% trypan blue, obtained by manual counting under inverted microscopy (Nikon, Tokyo, Japan). The relative percentage of parasite viability was defined as: (mean of the treated parasite/mean of the control) à 100. The lowest concentration of extract that inhibited 90% of A. castellanii growth was recorded as the MIC. The commercial antibiotic agent, chlorhexidine was used as positive control, while 1% DMSO was used as negative control.\n\nAnti-encystation was performed as previously studied11 with modifications. Briefly, Acanthamoeba trophozoites (5Ã105 cells/mL) were incubated in Neffâs medium in a 96-well plate containing Propolis extracts at different concentrations (1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC). Plates were incubated at 28°C for seven days, and the total amoebae number was counted using a hemocytometer (Boeco, Hamburg, Germany). Following the sodium dodecyl sulfate (SDS, 0.5% final concentration) was added and incubated for 1 hour to dissolve trophozoites and immature cyst. The remaining cysts were counted using a hemocytometer after the addition of SDS. To quantify encystation, the percentage of Acanthamoeba encystation was determined as follows: (total number of amoebae post-SDS treatment/total number of amoebae pre-SDS treatment) à 100. Phenylmethylsulfonyl fluoride (PMSF) (10 mM final concentration) was used as a positive control, whereas 1% DMSO was used as a negative control.\n\nFor excystation, Acanthamoeba cysts (5Ã105 cells/mL) were incubated with various concentrations of Propolis extracts (1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC) in PYG medium in 96-well plate at 28°C for seven days12. The effects of the extract on excystation were observed under an inverted microscope. The total amoebae were counted using a hemocytometer while SDS (0.5% final concentration) was added and incubated for 1 hour to dissolve trophozoites and immature cysts. The remaining cysts were counted after the addition of SDS. To quantify excystation, the percentage of Acanthamoeba excystation was determined as follows: (total number of amoebae pre-treatment with SDS â total number of amoebae post-SDS treatment)/total number of amoebae pre-SDS treatment) à 100. PMSF (10 mM final concentration) and amoebae alone were used as control.\n\nThe anti-adhesion assay was modified as previously reported13. Trophozoites (4 à 105 cells/mL) were added to each well of a 96-well polystyrene microtiter plate supplemented with 1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC of Propolis extract. Plates were incubated at 28°C without shaking for 24 hours. After incubation, a removing step to discard unbound trophozoites was performed. Plates were washed once with 0.1 M PBS, then air dried for 30 minutes at room temperature. The wells were stained with 0.1% crystal violet assay for 30 minutes. The crystal violet was eliminated, and the plates were washed with water and air dried. An aliquot of DMSO was added to the well and the absorbance was read at OD570 nm. Wells containing trophozoites without compounds or chlorhexidine were used as controls. The percentage of inhibition was calculated by following the formula: percentage of inhibition = (control OD â test OD/control OD) à 100.\n\nThe cytotoxic effects of the most active Propolis extract were evaluated using the Vero cell line (ECACC 84113001, RRID:CVCL_0059). Cells were cultured in Dulbeccoâs Modified Eagleâs medium (DMEM) (Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS (Sigma Aldrich, St. Louis, USA), and 1% antibiotic containing penicillin G (100 units/mL) and streptomycin (100 ÎŒg/mL). The culture was incubated at 37°C, humidified with 5% CO2 in an incubator (non-shaking). After the cells reached 90% confluence, the detachment was performed with trypsin and ethylene diamine tetra-acetic acid (EDTA) and incubated at 37°C in 5% CO2. Single cells at a density of 1.5 à 104 cells/100 ÎŒL were seeded into each well of a 96-well polystyrene plate and allowed to attach for 24 hours. Then, 100 ÎŒL propolis extract, eye drops, and combined set were gently added. After incubation for 24 hours, the cytotoxic effects were determined using an MTT assay14,15. The absorbance was measured using a microplate reader (Biotek, Cork, Ireland) at 570 nm. The survival percentage was calculated using the following equation:\n\nABt and ABu denote the absorbance values of treated and untreated cells, respectively.\n\nPropolis extract (20 mg/mL) was diluted in ethanol (1:10), the solution was centrifuged for 10 min at a speed of 10,000 rpm at temperature of 10°C. The solution was used for analysis. GC-MS analysis was performed using Agilent Technology 7890 A (GC) equipped with 5977A Mass Selective Detector (MS) (Agilent, California, USA). A VF-WAXms capillary column of dimensions 30 m à 250 à 0.25 ÎŒM was used with helium gas as the carrier at 30 m à 250 à 0.25 ÎŒM at a flow rate of 1 mL/min. The column temperature was initially programmed at 60°C, which was increased to 160°C at 10°C/minute and further increased to 325°C at 2.5°C/minute, hold time for 15 minutes. The mass spectra was collected at 70 eV ionization voltage over the range of m/z 35 to 500 in full scan mode. Chemical constituents were identified by comparing their mass spectral data with those from the Wiley library.\n\nThe experiments were repeated in triplicate. All data were recorded and entered into IBM SPSS Statistics (RRID:SCR_016479) version 26.0 (SPSS Inc. Chicago, IL, USA). Data were expressed as mean ± SD. Statistical analysis was conducted using a two-tailed unpaired Studentâs t-test. p < 0.05 was considered statistically significant in all analyses.\n\nThe effect of Propolis compounds on essential proteins of A. castellanii was investigated using the computational modelling method. This study focuses on three critical proteins: the Sir2 family protein, the mannose-binding protein, and the G protein-coupled receptor. The I-TASSER server was used to predict the 3D structures of these proteins16,17. FASTA sequences of A. castellanii Sir2 family protein (AcSir2) (NCBI Reference Sequence: XP 004358245.1)18, A. castellanii mannose-binding protein (AcMBP) (GenBank: AAT37865.1)19, and A. castellanii G protein-coupled receptor (AcGPCR) (GenBank: ELR16814.1)18 were used as inputs, with no constraints or applied templates. The most confidently predicted model was constructed using the most significant templates in the threading alignments. Then, the quality of the predicted 3D model was further improved using ModRefiner20. Finally, the stereochemical quality of the protein structures was determined using PROCHECK (RRID:SCR_019043)21.\n\nIn this study, we used molecular docking to measure the binding energies of major compounds of Propolis such as pinocembrin, chrysin, and tectochrysin to those of A. castellanii essential proteins such as AcSir2, AcMBP, and AcGPCR to identify potential protein targets. Prior to the molecular docking process, the protein structures were dehydrated to expose only amino acid residues. Then, polar hydrogens were assigned to the protein structure, nonpolar hydrogens were merged, and Kollman charges were added to amino acid residues. The partial charges and atom types were assigned to stabilized protein structures and saved the files in the PDBQT formats (Protein Data Bank (PDB), Partial Charge (Q), and Atom Type (T)). For the preparation of the ligand, the PubChem database was queried for the 3D structures of pinocembrin (PubChem CID: 68071)22, chrysin (PubChem CID: 5281607)23, and tectochrysin (PubChem CID: 5281954)24. Next, polar hydrogens and Gasteiger charges were introduced to the ligand structures, and nonpolar hydrogens were merged. Finally, the ligand structures were saved in the PDBQT format for stabilized ligand structures. After the receptor and ligand structures were prepared, the grid maps representing the system in the actual docking process were calculated with AutoGrid4 software version 4.2. The dimension of the grid was set to sufficiently cover the whole receptor structure (126 à 126 à 126 Ã
), with a spacing of 0.608 Ã
. All procedures were carried out using the AutoDock Auxiliary Tool (ADT) version 4.225,26.\n\nAutoDock4 version 4.225,26 was chosen for this purpose. Each docking step consisted of 50 GA runs with a maximum population size of 200 units. The total energy evaluation for each docking was 2,500,000 units. The average mutation rate was 0.02, the average cross-over rate was 0.80, and the average elitism value for each docking was 1. The Lamarckian Genetic Algorithm was used to combine local search (using the Solis and Wets algorithm) and global search (using the Genetic Algorithm alone)27. This parameter was used to perform 10,000 independent docking runs on each ligand. This step was repeated five times to ensure the results were accurate. The protein-ligand lowest binding energy (ÎGbind) and the inhibitor constant were determined using AutoDock Auxiliary Tool (ADT) version 4.225,26.\n\nMD simulations were performed using the Desmond module (RRID:SCR_014575) from Schrödinger suite (RRID:SCR_014879)28. In this process, hydrogen bonds were assigned according to standard procedures. The optimized potentials for liquid simulations (OPLS) force field was then applied to the protein and ligand complexes. The energy of the complexes was minimized after submerging them in a transferable intermolecular potential with 3 points (TIP3P) water model at a distance of 10 Ã
from the center of the box. The system was then neutralized by adding sodium and chloride ions, mimicking the in vivo environment. Molecular dynamic simulations were performed for 100 ns using ensembles of constant numbers of particles, pressure, and temperature (NPT) with a recording interval of 100 ps. The temperature was set to be 310.15 K and a pressure of roughly 1.01325 bar29,30.\n\nThe following formula was used to determine the root mean square deviation (RMSD) trajectories of the protein-ligand interaction:\n\n\n\nwhere N is the number of chosen atoms, r' is the position of the chosen atoms in a frame x after they have overlapped in the reference frame, where frame x is captured at time tx, and tref is the reference time. Each additional simulation frame required a new repeat of this process28.\n\nThe protein residues' root mean square fluctuation (RMSF) trajectories were determined using the following formula:\n\n\n\nwhere T stands for the trajectory time interval used to calculate the RMSF, râ² stands for the position of the atoms in residue I following superposition in the reference, ri stands for the position of residue I, tref stands for the reference time, and the angle brackets signify that the square distance is averaged on the atoms in the selected residue28.\n\nThe ligand atoms' RMSF trajectories were estimated using the following formula:\n\n\n\nwhere T is the trajectory time interval used to calculate the RMSF, r' is the position of atom I in the reference at time t following superposition on the reference frame, tref is the reference time, and r is the location of atom I in the reference at time tref28.\n\nDesmond Schrödinger's module's simulation interaction diagram tool was used to analyze protein-ligand interactions, protein-ligand RMSD, and protein and ligand RMSF28â30.\n\nThe Prime Molecular Mechanics Generalized Born Surface Area (MM-GBSA) approach31, which integrates the GBSA continuum solvent model32, was used to calculate the contributions of enthalpy and entropy-related components toward the binding of the ligand-protein complex. The contributions from molecular mechanics energies, polar solvation, and nonpolar solvation terms were estimated (kcal/mol) using the equation:\n\n\n\nWhere,\n\nÎGbind = Calculated binding free energy of complex\n\nGcomplex = Binding free energy of minimized complex\n\nGprotein = Binding free energy of receptor\n\nGligand = Binding free energy of unbound ligand\n\nThe proteins and ligands in this study were visualized using BIOVIA Discovery Studio version 21.1.0.20298 (RRID:SCR_015651) software33 and the Mol Viewer34.\n\nDrug-likeness profiles of ligands were unraveled through SwissADME, a free web tool to evaluate pharmacokinetics, drug-likeness, and medicinal chemistry of small molecules35.\n\nThe pharmacokinetic properties of the ligands, such as chemical absorption, distribution, metabolism, excretion, and toxicity (ADMET), were analyzed using the pkCSM ADMET descriptors algorithm methodology, an approach to the prediction of pharmacokinetic properties that relies on graph-based signatures36. In brief, the canonical SMILES of the ligands (pinocembrin, chrysin, and tectochrysin) acquired from the PubChem database (RRID:SCR_004284) were used for input data, and ADMET profiles were generated. The Caco-2 permeability, intestinal absorption (human), and skin permeability were estimated to predict the absorption level of the ligands. The steady-state volume of distribution (VDss), fraction unbound (human), blood-brain barrier (BBB) permeability, and central nervous system (CNS) permeability were evaluated to predict the distribution of the ligands in various tissues. To predict the metabolism of the ligands in the human body, the ligands were determined whether they are likely to be CYP2D6/CYP3A4 substrates (the two main subtypes of cytochrome P450) or Cytochrome P450 inhibitors or not. To predict the excretion of the ligands, total compound clearance was measured. The compounds also determined whether they are likely going to be renal organic cation transporter 2 (OCT2) substrates or not. Finally, the toxicity of the ligands was predicted by AMES toxicity, hERG I/II inhibitor, oral rat acute toxicity (LD50), oral rat chronic toxicity (LOAEL), hepatotoxicity, skin sensitization, and Minnow toxicity.\n\n\nResults\n\nThe effect of Propolis extracts on both strains of Acanthamoeba was examined, and the results exhibited as MIC are presented in Table 137. Propolis extracts from the Kermanshah city exhibited the most inhibitory activity. The values of the minimum inhibitory concentration (MIC) ranged from 62.5 to 125 µg/mL in trophozoite form. But this extract had no inhibitory activity in cyst form at 1,000 µg/mL concentration. In the positive control, chlorhexidine exhibited MIC values of 8 to 16 and 32 to 64 µg/mL for trophozoite and cyst form, respectively. Therefore, Propolis from Kermanshah city was chosen for further study.\n\nMIC: minimal inhibitory concentration. ATCC: American Type Culture Collection.\n\nTo assess the effect of Propolis extract on A. castellanii encystation, PMSF was used as a positive control in Neffâs medium. According to the data presented in Figure 1, the results revealed that the Propolis-Kermanshah city exhibited inhibition of A. castellanii encystation at all concentrations. The formation of mature cysts significantly reduced after Propolis extract treatment at 1/16 MIC on both strains of A. castellanii ATCC50739 (21%) (Figure 1A) and A. castellanii ATCC30010 (17%) (Figure 1B). In the 5 mM PMSF group, encystation was reduced to 6% and 8% for ATCC50739 and ATCC30010, respectively.\n\nPropolis extract reduced the encystation in a dose-dependent manner on (A) A. castellanii ATCC50739 and (B) A. castellanii ATCC30010. The experiments were repeated three times, and the average values are presented with error bars representing standard deviations. *; significantly different at a P value of <0.05 by Studentâs t test. PMSF, phenylmethylsulfonyl fluoride; ATCC, American Type Culture Collection.\n\nThe effect of Propolis extract treatment on excystation was assessed in PYG medium. The excystation rate decreased to 44% and 42% after exposure to high concentrations of propolis extract at 1/2 MIC of trophozoites (Figure 2A and B). PMSF significantly inhibited Acanthamoeba excystation (13% and 4%) at 5 mM concentration.\n\nPropolis extract reduced the excystation on (A) A. castellanii ATCC50739 and (B) A. castellanii ATCC30010. The experiments were repeated three times, and the average values are presented with error bars representing standard deviations. *; significantly different at a P value of <0.05 by Studentâs t test. PMSF, phenylmethylsulfonyl fluoride; ATCC, American Type Culture Collection.\n\nTo evaluate the influence of Propolis extract on the adhesion properties of Acanthamoeba trophozoites, the adhesion of trophozoites to the plastic surface varied and depended on the concentrations of extract. The strongest anti-adhesion was observed in trophozoites treated with 1/2 MIC concentration of extract (Table 2)37 in both strains of Acanthamoeba when compared with the untreated control.\n\nMIC: minimal inhibitory concentration. ATCC: American Type Culture Collection.\n\nAfter 24 hours of treatment with Propolis extract, the number of viable cells was constant at low concentrations, ranging from 8â64 µg/mL. However, the survival rate of Vero cells was lower when treated with the extract at concentrations of 128â1,000 µg/mL.\n\nThe GC-MS analysis of the Propolis-Kermanshah city extract allowed the identification of 52 compounds. Chrysin (18.86%) was the main compound present in the Propolis extract, followed by pinocembrin (15.02%), and tectochrysin (9.88%), respectively.\n\nThe optimal 3D structural models of AcSir2, AcMBP, and AcGPCR were constructed using I-TASSER server and the top 10 threading templates. Then, the best C-score model was selected and refined. The refined 3D structure models of AcSir2, AcMBP, and AcGPCR are illustrated in Figure 338,39. The AcSir2 is a protein located inside the nucleus. The protein consists of 536 amino acids (aa) that contain the SIR2 super-family region (aa residues 36â297) and YEATS family region (aa residues 443-524). The AcMBP is a large protein located at the cell membrane. The protein consists of 833 aa, and some residues such as aa residues 732-760 are transmembrane proteins. The AcGPCR is a protein also located in the cell membrane. The protein consists of 456 aa, and some of them are transmembrane proteins, such as aa residues 182-202, 214-236, 248-274, 286-305, 311-332, 353-375, and 381-401. The protein contains the lung seven-transmembrane receptor region (aa residues 140-413). The stereochemical quality of protein structures was analyzed using PROCHECK. The Ramachandran plot of the AcSir2 model identified 71.0% of the residues in the most favored regions, 25.8% of the residues in the other allowed regions, and only 3.2% of the residues in disallowed regions. The Ramachandran plot of the AcMBP model discovered 64.7% of the residues in the most favored regions, 32.3% of the residues in the other allowed regions, and only 3.0% of the residues in disallowed regions. The AcGPCR model's Ramachandran plot determined 85.5% of the residues in the most favored regions, 13.3% in other allowed regions, and only 1.2% in disallowed regions.\n\nAcSIR2: blue represents the SIR2 superfamily region, yellow represents the YEATS family region. AcMBP: Orange represents transmembrane proteins; green represents a domain of an unknown function. AcGPCR: purple represents Lung seven-transmembrane receptor. AcSIR2, A. castellanii Sir2 family protein; AcMBP, A. castellanii mannose-binding protein; AcGPCR, A. castellanii G protein-coupled receptor.\n\nThe molecular docking of Propolis compounds such as: pinocembrin, chrysin, and tectochrysin against three essential proteins of A. castellanii was performed using AutoDock 4. The results are illustrated in Figure 4âFigure 639,40. The Pinocembrin demonstrated good binding potential to the AcSir2 with binding energy (ÎGbind) of -7.63 kcal/mol and the inhibitory constant (Ki) of 2.57 µM. The compound interacts with the residues Glu147 through the conventional hydrogen bond (H-bond), Thr476 through Pi-lone pair, Phe477 through Pi-Pi T-shaped, Ser478 through conventional H-bond and Pi-Lone Pair, and Val482 through conventional H-bond. Chrysin exhibited a high affinity for AcSir2, with a ÎGbind of -8.05 kcal/mol and a Ki of 1.26 µM. The compound interacts with the residues Glu147 via the conventional H-bond, Phe272 via Pi-Pi T-shaped, Thr476 via Pi-lone pair, and Ser478 via Pi-donor H-bond. Tectochrysin exhibited an excellent affinity for AcSir2, with a ÎGbind of -8.12 kcal/mol and a Ki of 1.12 µM. The compound interacts with residues Arg122 through carbon or Pi-donor H-bond, Leu123 through alkyl or Pi-alkyl, Gly124 through conventional H-bond, Ile269 through alkyl or Pi-alkyl, Phe272 through Pi-Pi T-shaped, Phe477 through Pi-Pi T-shaped, and alkyl or Pi-alkyl, Ser478 through Pi-lone pair, and carbon or Pi-donor H-bone, and Pro479 through alkyl or Pi-alkyl (Figure 4).\n\n(A) Binding site of the ligands toward the AcSIR2 protein, purple compound represents pinocembrin, red compound represents chrysin, yellow compound represents tectochrysin. (B) A schematic representation of the detailed interactions of the ligand atoms with the protein residues. (C) Binding affinity and inhibitory constant prediction of propolis compounds against Sir2 family protein of Acanthamoeba castellanii. AcSIR2, A. castellanii Sir2 family protein.\n\n(A) Binding site of the ligands toward the AcMBP protein, purple compound represents pinocembrin, red compound represents chrysin, yellow compound represents tectochrysin. (B) A schematic representation of the detailed interactions of the ligand atoms with the protein residues. (C) Binding affinity and inhibitory constant prediction of propolis compounds against mannose-binding protein of Acanthamoeba castellanii. AcMBP, A. castellanii mannose-binding protein.\n\n(A) Binding site of the ligands toward the AcSIR2 protein, purple compound represents Pinocembrin, red compound represents Chrysin, yellow compound represents Tectochrysin. (B) A schematic representation of the detailed interactions of the ligand atoms with the protein residues. (C) Binding affinity and inhibitory constant prediction of propolis compounds against G protein-coupled receptor of Acanthamoeba castellanii. AcGPCR, A. castellanii G protein-coupled receptor; AcSIR2, A. castellanii Sir2 family protein.\n\nPinocembrin demonstrated a weak binding affinity for AcMBP with ÎGbind of -6.34 kcal/mol and the Ki of 22.62 µM. The compound interacts with residues Pro593 through conventional H-bond and Pi-alkyl, Cys610 through Pi-sulfur, Thr625 through Pi-sigma, and Cys632 through Pi-alkyl. Chrysin had a very low affinity for AcMBP, with ÎGbind of -6.15 kcal/mol and the Ki of 30.92 µM. The compound interacts with the residues Pro263, Val267, and Pro317 through Pi-alkyl; Cys327 through Pi-sulfur and Pi-alkyl; Asp366 and Asn367 through conventional H-bond; and Phe369 through Pi-Pi Stacked. Tectochrysin showed a low affinity for AcMBP, with ÎGbind of -6.32 kcal/mol and the Ki of 23.21 µM. The compound interacts with residues Pro593 and Pro594 through Conventional H-bond; Glu596 through Pi-sigma; Cys610, Cys627, and Cys632 through Pi-sulfur; and Cys612 and Cys632 through Pi-Alkyl (Figure 5).\n\nPinocembrin demonstrated a weak binding affinity for the AcGPCR with ÎGbind of -7.04 kcal/mol and the Ki of 6.96 µM. The compound interacts with residues Ile275 through Pi-alkyl, Phe278 through conventional H-bond and unfavorable donor-donor interaction, Leu279 through Pi-alkyl, Asp283 through Amide-Pi stacked, Lys284 through Pi-alkyl, and Arg346 through conventional H-bond. With a ÎGbind of -6.98 kcal/mol and Ki of 7.7 µM, chrysin exhibited a low affinity for AcGPCR. The compound interacts with AcGPCR in the same way as the pinocembrin-AcGPCR complex, except for the residue Phe278, whose conventional H-bonding did not occur for this compound. Finally, tectochrysin exhibited a low affinity for AcGPCR, with ÎGbind of -7.17 kcal/mol and the Ki of 5.51 µM. The compound interacts with AcGPCR the same as the pinocembrin-AcGPCR complex, except for the residue Phe278 in which there was no interaction for this compound (Figure 6). Based on the molecular docking result, pinocembrin, chrysin, and tectochrysin demonstrated inhibition potential towards the AcSir2 protein. Tectochrysin showed the most robust inhibition, followed by chrysin and pinocembrin. Thus, the molecular dynamics of these complexes were then simulated to understand the dynamic motions and analyze the stabilities of these protein-ligand complexes.\n\nThe dynamic motions of the apo and docked complexes were further analyzed by molecular dynamic simulations at 100 ns using the Desmond module of Schrödinger's suite. The results of MD simulations of the apo and bound forms of AcSir2 protein are illustrated in Figure 7 and Figure 839,41, respectively. For an MD run of 100 ns, the RMSD and the RMSF were predicted for the apo and bound forms. A ligandâs interaction can ward off unfolding and stabilize the protein42. Hence, we analyzed the proteinâs secondary structures before and after docking to understand the conformational changes due to ligand binding. The RMSD quantifies the average change in displacement of a selection of atoms relative to a reference frame for a particular frame. Figure 7A, Figure 8A, 8D, and 8G demonstrated the protein RMSD from the simulation of the AcSir2 apo form, pinocembrin-AcSir2 complex, chrysin-AcSir2 complex, and tectochrysin-AcSir2 complex, respectively. The Protein RMSD (P-RMSD) shows how the RMSD of a protein has changed over time (left Y-axis). After aligning all of the protein frames with the backbone of the reference frame, the atoms are chosen to figure out the P-RMSD. During the simulation, the calculation of the P-RMSD can give information about how the structure is built. For the ligand RMSD (L-RMSD), the L-RMSD value (right Y-axis) shows how stable the ligand is concerning the protein and its binding pocket. 'Lig fit Prot' illustrated the RMSD of a ligand after the protein-ligand complex was aligned in the reference protein backbone and the RMSD of the ligand heavy atoms was determined. If the observed values exceed the P-RMSD by a significant amount, the ligand almost certainly has diffused away from its initial binding site. The mean values of P-RMSD of the apo-AcSir2 was around the 10 Ã
(Figure 7A). The P-RMSD values of the Pinocembrin-AcSir2 complex wildly deviated at around the first 12 ns. After that, the fluctuation was regular at the end of the simulation. An average of RMSD values is stable after 12 ns, indicating that the system has equilibrated during this simulation. Furthermore, for the L-RMSD values of the Pinocembrin-AcSir2 complex, the observed values are significantly lower than the P-RMSD, so the ligand has likely fixed in its initial binding site (Figure 8A, Supplementary video 1 found as Underlying data43). The P-RMSD values of the chrysin-AcSir2 complex deviated from 0 to 14 Ã
in the first duration of 18 ns and slightly decreased during 18 ns to 50 ns. The fluctuation was regular at the end of the simulation after 70 ns, with the highest point about 15 Ã
and the lowest point about 10 Ã
. An average of RMSD values is constant after 70 ns, indicating that the system has equilibrated during this simulation. The L-RMSD values of this complex show significantly lower than the RMSD of the protein during the first 90 ns. However, the values dramatically increase around 90 ns and are higher than the P-RMSD. Almost obviously, the ligand may have diffused away from its initial binding site (Figure 8D, Supplementary video 2 found as Underlying data43). The fact that the P-RMSD values of the tectochrysin-AcSir2 complex strongly deviated throughout the simulation shows that a substantial conformational change has occurred in the protein. However, the overall average is relatively stable after 60 ns, indicating that the system has equilibrated during this simulation. The L-RMSD values of this complex are lower than the RMSD of the protein during the first 80 ns. However, the values gradually increase during the simulation time of 80â90 ns, then more extensive than the P-RMSD at approximately 90 ns. Clearly, the ligand may have diffused away from its initial binding site (Figure 8G, Supplementary video 3 found as Underlying data43). The RMSF can characterize local changes in the protein chain and the positions of the ligand atoms. Figure 7B, Figure 8B, 8E, and 8H demonstrated the protein and ligand RMSF from the simulation of the apo-AcSir2, pinocembrin-AcSir2 complex, chrysin-AcSir2 complex, and tectochrysin-AcSir2 complex, respectively. For the protein RMSF (P-RMSF), the peaks in this plot correspond to the protein regions that fluctuate the most during the simulation. The P-RMSF of the apo-AcSir2 strongly fluctuated at amino acid residues Pro19, Pro112, Cys192-Gly212, Pro318-Ala357, Arg364-Met378, Thr409-Glu412, Pro423, His432, Ala434-Pro436, and Pro515-Ala536 (Figure 7B). The P-RMSF of the pinocembrin-AcSir2 complex strongly fluctuated at amino acid residues Pro112, Pro204, Asp317, Pro320, Pro324, Pro334, Pro423, Pro436, Val450, and Pro515. However, these residues were not ligand contacts residues (Figure 8B). The P-RMSF of the chrysin-AcSir2 complex immensely fluctuated at amino acid residues Pro19, Pro112, Pro204, Asp317, Pro324, Pro334, Pro423, His432, Pro436, Lys451, and Pro515. These residues were also not ligand contacts residues (Figure 8E). The P-RMSF of the tectochrysin-AcSir2 complex wildly fluctuated at amino acid residues Pro19, Pro112, Pro204, Val319, Pro324, Pro423-Thr437, Thr443, Pro151, and Gly525. Almost all these residues were not ligand contacts residues, except for the Pro204 position (Figure 8H). The result illustrated that the binding of a ligand can prevent protein unfolding and stabilize it. During the simulation, the interactions of the protein with the ligand can be monitored. The protein-ligand contacts diagrams for the pinocembrin-AcSir2 complex, chrysin-AcSir2 complex and tectochrysin-AcSir2 complex are illustrated in Figures 8C, 8F, and 8I, respectively. The stacked bar charts demonstrated that all complexes exhibited H-bonds, hydrophobic interactions, ionic bonds, and water bridges during the simulation. The PRIME MM-GBSA binding free energy values of the ligand-AcSir2 complexes are given in Table 3.\n\n(A) Plot of the P-RMSD of the apo-AcSIR2 protein. (B) Plot of the P-RMSF of the apo-AcSIR2 protein. P-RMSD, protein root mean square deviation; P-RMSF, protein root mean square fluctuation; AcSIR2, A. castellanii Sir2 family protein.\n\n(AâC) Simulation interactions diagram of pinocembrin-AcSIR2 complex. (A) Plot of protein-ligand RMSD. (B) Plot of protein RMSF. (C) Histogram of protein-ligand contacts categorized by type of interactions: hydrogen bonds (green), hydrophobic (purple), ionic (magenta), and water bridges (blue). (D-F) Simulation interactions diagram of chrysin-AcSIR2 complex. (D) Plot of protein-ligand RMSD. (E) Plot of protein RMSF. (F) Histogram of protein-ligand contacts categorized by type of interactions: hydrogen bonds (green), hydrophobic (purple), ionic (magenta), and water bridges (blue). (GâI) Simulation interactions diagram of tectochrysin-AcSIR2 complex. (G) Plot of protein-ligand RMSD. (H) Plot of protein RMSF. (I) Histogram of protein-ligand contacts categorized by type of interactions: hydrogen bonds (green), hydrophobic (purple), ionic (magenta), and water bridges (blue). RMSD, root mean square deviation; RMSF, root mean square fluctuation; AcSIR2, A. castellanii Sir2 family protein.\n\n1Binding free energy, 2Coulombic energy, 3Hydrogen bond energy, 4Lipophilic energy, 5Van der Waal energy. MM-GBSA: Molecular Mechanics Generalized Born Surface Area. AcSIR2: A. castellanii Sir2 family protein.\n\nAfter careful analysis of the drug-likeness properties of the ligands using SwissADME, the result indicated that the compound properties are within the range of drug-likeness based on various filters such as Lipinski44, Ghose45, Veber46, Egan47, and Muegge48 (Table 4).\n\nThe ADMET properties of the pinocembrin, chrysin, and tectochrysin are presented in the Table 5. To predict the absorption level of the compounds, water solubility, Caco-2 permeability, intestinal absorption (human), and skin permeability were estimated. A compound is easy to absorb if Caco-2 permeability is high. The Caco-2 permeability is considered as high if it has an apparent permeability coefficient (Papp) > 8 à 10-6 cm/s (or log Papp > 0.90). The results showed that all ligands were predicted to have high Caco-2 permeability. About the human intestinal absorption prediction, a compound is poorly absorbed if absorbance is less than 30%. The results proved that all compounds were considered to have a good absorption. With regards to skin permeability, if a compound has a logKp > -2.5, the compound is predicted to have a relatively low skin permeability. The results indicated that all compounds were predicted to have good skin permeability.\n\nVDss: the steady state volume of distribution. BBB: blood-brain barrier. CNS: central nervous system. AMES: assay of the ability of a chemical compound to induce mutations in DNA. OCT2: organic cation transporter 2.\n\nTo predict the distribution of the compounds in various tissues, the VDss, Fraction unbound (human), BBB permeability, and central CNS permeability were evaluated. The VDss is relatively low if lower than 0.71 L/kg (log VDss < -0.15). Whereas it is high if higher than 2.81 L/kg (log VDss > 0.45). The results demonstrated that the VDss of the pinocembrin was higher than the chrysin and tectochrysin. The higher the VD is, the more of a ligand is distributed in tissue rather than plasma, as demonstrated in the pinocembrin. With regards to BBB and CNS permeability, the results indicated that the pinocembrin readily crossed the BBB, while chrysin and tectochrysin might penetrate the CNS.\n\nAs cytochrome P450 is responsible for the metabolism of many drugs in liver, to predict metabolism of compounds, the compounds were determined whether they are likely to be CYP2D6/CYP3A4 substrates (the two main subtypes of cytochrome P450) or Cytochrome P450 inhibitors or not. The result predicted that all compounds were not substrates and inhibitors of the CYP2D6. However, all of them were inhibitors of CYP1A2, CYP2C19, and CYP2C9. Moreover, the tectochrysin was a substrate and inhibitor of CYP3A4.\n\nTo predict the excretion of the compounds, total compounds clearance was measured. It was also determined whether the compounds were likely going to be renal organic cation transporter 2 (OCT2) substrates or not. With regards to total compounds clearance, the total clearance of the tectochrysin is the highest, followed by chrysin, and pinocembrin. Remarkably, all compounds were not predicted to be renal OCT2 substrates.\n\nFinally, to predict the toxicity of the compounds, AMES toxicity, hERG I/II inhibitor, oral rat acute toxicity (LD50), oral rat chronic toxicity (LOAEL), hepatotoxicity, skin sensitization, and Minnow toxicity were predicted. Notably, the results predicted that none of the compounds were mutagenic or hERG I/II inhibitors, and none of them showed hepatotoxicity or skin sensitization.\n\n\nDiscussion\n\nThe pharmaceutical activities of natural products have historically been screened because they are thought to have key roles in drug discovery, are inexpensive and rarely have undesirable side effects. Propolis has been known for a long time and attracted scientific interest due to its biological activities such as anti-viral, anti-bacterial, anti-fungal, anti-protozoal, anesthetic, antioxidant, anti-tumoral, anti-cancer, anti-hepatotoxic, anti-mutagenic, anti-septic and anti-inflammatory activities, in addition to being utilized for its cytotoxic activity49,50. In vitro studies of its antiparasitic effect were reported against Leishmania spp., Trypanosoma spp., Plasmodium spp., Cryptosporidium spp., Giardia spp., Toxoplasma gondii, Trichomonas vaginalis, and Blastocystis spp.51. In literature, Propolis extract has reported amoebistatic activity between 2.0 and 6.0 mg/mL and its effects were amoebicidal at 8.0 mg/mL or higher52. Here, the anti-Acanthamoeba activities of three Propolis extracts from different cities in Iran were screened. The highest activity was obtained from the Propolis ethanolic extract of Kermanshah city, and the MIC against trophozoites was 62.5 µg/mL and 125 µg/mL for A. castellanii ATCC30010 and ATCC50739, respectively. Propolis composition included more than 180 different types of chemicals53 depending on several factors such as extraction method, source of plant, season, and local flora54. Flavonoid compounds like chrysin and pinocembrin are commonly identified in Romanian, Turkish and Polish propolis. The type of Uruguayan propolis mentioned other flavonoid compounds like tectochrysin, galangin and kaempferol55. This study revealed the main compounds of Propolis from Kermanshah city were chrysin, pinocembrin, and tectochrysin. To determine the cytotoxic effect of the extract at a concentration of at least 0.128 mg/mL was demonstrated against Vero cells. Our data agreed with Vural et al.56, in which the Propolis concentration at higher than 7.81 mg/mL caused corneal epithelial cell damage. The safe concentration of Propolis at 1.4 mg/kg per day was also recommended57.\n\nA. castellanii ATCC50739 and ATCC30010 were tested for their encystment capability in Neffâs medium. The results seemed to encyst and presented the mature cysts in both media for seven days. The process of Acanthamoeba is an essential for the survival under unfavorable conditions58. The double wall of the Acanthamoeba cyst is resistant to many drugs and chemicals and leads to clinical drug resistance59. As only single cyst surviving in the cornea stroma after initial successful treatment, they can regularly excyst and lead to reinfection60. Thus, the inhibition of encystation process during the treatment of Acanthamoeba infections can lead to more favorable outcomes and enhances the potential of Acanthamoeba keratitis treatment. In this study, PMSF was used as a positive control to block serine proteinase, providing a significant inhibition of encystation. The data were in agreement with the results of Leitsch et al.61 in which the PMSF inhibited the proteolytic activity at the early stage of encystation. Propolis extract at low concentration (1/16 MIC) was able to inhibit the encystation of A. castellanii ATCC50739 and ATCC30010. The low concentration caused a reduction in the level of encystation of around 80â90%. The high concentrations (1/2-1/8 MIC) gave a < 20% reduction in the encystment levels, which suggests that low concentration of Propolis extract is suitable for inhibiting the encystment process. The main mechanism underlying inhibition of encystation by Propolis remain largely unknown. Aqeel et al.62 mentioned phenolic compounds such as resveratrol and demethoxycurcumin are strong antioxidants with Acanthamoeba growth inhibitory effects in vitro. It raises the possibility that antioxidant activity may be required to inhibit Acanthamoeba encystation. Furthermore, Mahboob et al.63 reported that other phenolic compounds i.e., ester of caffeic acid and quinic acid, demonstrated the inhibitory effect on encystation by scavenging reactive oxygen species within Acanthamoeba cytoplasm.\n\nThe use of therapeutic agents for Acanthamoeba infection may lead to cyst formation, a drug-resistance stage, and transformation of cysts to trophozoites that lead to recurrence of infection64. The fluids or some microorganisms in eye infection may provide an appropriate condition to induce excystation of surviving Acanthamoeba cyst65. This reason remains a challenge for Acanthamoeba keratitis prevention. Although Propolis extract from Kermanshah city did not inhibit the growth of Acanthamoeba cysts at 1,000 µg/mL, it exhibited excystment inhibition at 62.5 and 31.25 µg/mL. This evidenced that it prevented the recurrence of infection because there was no change of morphological transformation from cysts to trophozoites. However, it remains unclear on how Propolis inhibited Acanthamoeba excystation. Maslinic acid, a natural triterpene found in olives and Propolis, has been shown to inhibit parasitic proteases enzymes66. These proteases enzymes are normally secreted within the first 24 hours, which may indicate an important role of the enzyme in excystation67.\n\nThe first step in the pathogenesis of Acanthamoeba infection is the adhesion to the surface of the host tissues. Subsequently, the adhesion to host cells, Acanthamoeba produce proteinase enzymes that work in concert to produce a potent cytopathic effect (CPE) involving killing of the host cells, degradation of epithelial basement, and penetration into the deeper layers of the cornea68. In this study, we showed that Propolis possess anti-amoebic properties and the capability to reduce amoebae adhesion on plastic plate. The highest adhesion was noticed in the control group, which was an untreated agent. Similar results were obtained in the current study, where anti-adhesion was observed in plastic plate and contact lenes belonging to Curcuma longa extract69, Annona muricata and Combretum trifoliatum extracts70, and Garcinia mangostana and their pure compounds13.\n\nBased on our results, we recognize the importance of developing Propolis extract to eliminate or inhibit the pathogenicity of Acanthamoeba. Therefore, the determination the main target of the pathogen in silico has been studied. A molecular docking simulation was carried out to investigate the binding affinities of the major compounds (chrysin, pinocembrin and tectochrysin) from the Propolis extract and essential proteins in Acanthamoeba (AcSir2, AcMBP, and AcGPCR). AcSir2 was classified as a class-IV sirtuin. This protein exhibited functional SIRT deacetylase activity, localized mainly in the nucleus, and its transcription was upregulated during encystation71. Acanthamoeba mannose-binding protein (AcMBP) is a virulence factor of the free-living amoeba, which is important for adhesion of the pathogen72. G proteins and GPCRs are well known key regulators of cellular communication and cellular functions including cell cycle, mitosis, and proliferation73.\n\nOur study revealed the potential capability of the pinocembrin, chrysin, and tectochrysin complex to form hydrogen bonds with the AcSir2 protein. The low binding energy indicates strong interactions between the compounds and AcSir2 protein. Sirtuins have been classified into five major classes (I, II, III, IV and V) and conserved from bacteria to humans71. In some parasites, Sir2 is located mainly in the nucleus and plays a role in cell function, proliferative life span and development under various conditions74. Notably, the regulation of AcSir2 expression is essential for growth and encystation in A. castellanii. In AcSir2-overexpressing encysting cells, the transcription of cellulose synthase was highly upregulated compared to control cells71. Moreover, MD simulations indicated that pinocembrin, chrysin, and tectochrysin can interact with AcSir2 protein. Chrysin and tectochrysin may have a probability of diffusing away from their initial binding site. Over the 100 ns of MD simulations, only the pinocembrin remained fixed within its initial binding site. Thus, this compound may be an excellent candidate for future anti-Acanthamoeba drug development. In this study, our successful combination of computational approaches and phenotypic screening led to the identification of compounds with noteworthy activities against Acanthamoeba.\n\n\nConclusions\n\nNatural products are one of the essential resources for drug discovery. Considering the pharmacological activities of Propolis extract against Acanthamoeba, its therapeutic potential should be considered. Our study was conducted with extract of Propolis. Moreover, molecular docking was used as a computational and easily accessible method to propose a binding mode of chrysin, tectochrysin and pinocembrin on a protein target. Molecular docking stimulation indicated that pinocembrin is the strongest binding site on AcSir2 protein. This noteworthy data further allows us to simulate the effects of pinocembrin or its synthetic structural modifications to optimize desirable activities and targets. Nevertheless, our results provide the possibility of finding a new series of anti-Acanthamoeba compounds that can act in combination with conventional drugs as an alternative therapeutic strategy for the treatment of AK.",
"appendix": "Data availability\n\nNCBI Protein: transcriptional regulator, Sir2 family protein [Acanthamoeba castellanii str. Neff]. Accession number XP_004358245.1; https://identifiers.org/ncbiprotein:xp004358245.118.\n\nNCBI Protein: mannose-binding protein [Acanthamoeba castellanii]. Accession number AAT37865.1; https://identifiers.org/ncbiprotein:AAT37865.119.\n\nNCBI Protein: G protein coupled receptor, putative [Acanthamoeba castellanii str. Neff]. Accession number ELR16814.1; https://identifiers.org/ncbiprotein:ELR16814.118.\n\nNCBI PubChem Compound: Pinocembrin. PubChem CID 68071; https://identifiers.org/pubchem.compound:6807122.\n\nNCBI PubChem Compound: Chrysin. PubChem CID 5281607; https://identifiers.org/pubchem.compound:528160723.\n\nNCBI PubChem Compound: Tectochrysin. PubChem CID 5281954; https://identifiers.org/pubchem.compound:528195424.\n\nFigshare: In vitro RAW DATA.xlsx. https://doi.org/10.6084/m9.figshare.2121356337.\n\nFigshare: MD simulations Movie.rar. https://doi.org/10.6084/m9.figshare.2121356043.\n\nThis project contains the following underlying data:\n\n- Supplementary videos 1-3\n\nFigshare: Raw data for Molecular docking. https://doi.org/10.6084/m9.figshare.2121407940.\n\nThis project contains the following underlying data:\n\nâ Molecular docking result between Propolis compounds and A. castellanii G-protein coupled receptor (AcGPCR)\n\n- Pinocembrin (PubChem CID: 68071) docking with AcGPCR\n\n- Chrysin (PubChem CID: 5281607) docking with AcGPCR\n\n- Tectochrysin (PubChem CID: 5281954) docking with AcGPCR\n\nâ Molecular docking result between Propolis compounds and A. castellanii mannose-binding protein (AcMBP)\n\n- Pinocembrin (PubChem CID: 68071) docking with AcMBP\n\n- Chrysin (PubChem CID: 5281607) docking with AcMBP\n\n- Tectochrysin (PubChem CID: 5281954) docking with AcMBP\n\nâ Molecular docking result between Propolis compounds and A. castellanii Sir2 family protein (AcSir2)\n\n- Pinocembrin (PubChem CID: 68071) docking with AcSir2\n\n- Chrysin (PubChem CID: 5281607) docking with AcSir2\n\n- Tectochrysin (PubChem CID: 5281954) docking with AcSir2\n\nFigshare: Raw data for Molecular dynamics (MD) simulation. https://doi.org/10.6084/m9.figshare.2121416041.\n\nThis project contains the following underlying data:\n\nâ AcSir2-pinocembrin (PubChem CID: 68071) complex\n\nâ AcSir2-chrysin (PubChem CID: 5281607) complex\n\nâ AcSir2-tectochrysin (PubChem CID: 5281954) complex\n\nâ AcSir2 only\n\nFigshare: Raw data for the prediction of the three-dimensional structures. https://doi.org/10.6084/m9.figshare.2121418438.\n\nThis project contains the following underlying data:\n\nâ A. castellanii Sir2 family protein (AcSir2)\n\n- AcSir2 protein sequence\n\n- Result of 3D structure prediction from I-Tasser\n\nâ A. castellanii G-protein coupled receptor (AcGPCR)\n\n- AcGPCR protein sequence\n\n- Result of 3D structure prediction from I-Tasser\n\nâ A. castellanii mannose-binding protein (AcMBP)\n\n- AcMBP protein sequence\n\n- Result of 3D structure prediction from I-Tasser\n\nFigshare: F1000_raw figures. https://doi.org/10.6084/m9.figshare.2131229739.\n\nThis project contains the following underlying data:\n\n- Figure 3, Figure 4A, Figure 4B, Figure 4C, Figure 5A, Figure 5B, Figure 5C, Figure 6A, Figure 6B, Figure 6C, Figure 7, Figure 8A, Figure 8B, Figure 8C, Figure 8D, Figure 8E, Figure 8F, Figure 8G, Figure 8H, Figure 8I\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe highly appreciate the support of the Research Institute of Health Science (RIHS) at Walailak University for the laboratory facilities.\n\n\nReferences\n\nKot K, Åanocha-Arendarczyk N, Kosik-Bogacka D: Immunopathogenicity of Acanthamoeba spp. in the brain and lungs. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nClarke M, Lohan AJ, Liu B, et al.: Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling. Genome Biol. [Dataset]. 2013; 14(2): R11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarate M, Cao Z, Bateman E, et al.: Cloning and characterization of a novel mannose-binding protein of Acanthamoeba. J Biol Chem. [Dataset]. 2004; 279(28): 29849â56. PubMed Abstract | Publisher Full Text\n\nXu D, Zhang Y: Improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization. Biophys J. 2011; 101(10): 2525â2534. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaskowski RA, MacArthur MW, Moss DS, et al.: PROCHECK: A program to check the stereochemical quality of protein structures. J Appl Cryst. 1993; 26(2): 283â291. Publisher Full Text\n\nNational Center for Biotechnology Information: PubChem Compound Summary for CID 68071, Pinocembrin. [Dataset]. 2022. https://pubchem.ncbi.nlm.nih.gov/compound/Pinocembrin\n\nNational Center for Biotechnology Information: PubChem Compound Summary for CID 5281607, Chrysin. [Dataset]. 2022. https://pubchem.ncbi.nlm.nih.gov/compound/Chrysin\n\nNational Center for Biotechnology Information: PubChem Compound Summary for CID 5281954, Tectochrysin. [Dataset]. 2022. https://pubchem.ncbi.nlm.nih.gov/compound/Tectochrysin\n\nMorris GM, Huey R, Lindstrom W, et al.: AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. 2009; 30(16): 2785â2791. PubMed Abstract | Publisher Full Text | Free Full Text\n\nForli S, Huey R, Pique ME, et al.: Computational protein-ligand docking and virtual drug screening with the AutoDock suite. Nat Protoc. 2016; 11(5): 905â919. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSolis FJ, Wets RJB: Minimization by random search techniques. Math Oper Res. 1981; 6(1): 19â30. Publisher Full Text\n\nShaw DE: Desmond Molecular Dynamics System, Tools for Maestro-Desmond interoperability. Schrödinger, New York, 2021.\n\nPant S, Singh M, Ravichandiran V, et al.: Peptide-like and small-molecule inhibitors against COVID-19. J Biomol Struct Dyn. 2021; 39(8): 2904â2913. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThangavel N, Bratty MA, Al Hazmi HA, et al.: Molecular docking and molecular dynamics aided virtual search of OliveNet⢠directory for secoiridoids to combat SARS-CoV-2 infection and associated hyperinflammatory responses. Front Mol Biosci. 2021; 7: 627767. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShivakumar D, Williams J, Wu Y, et al.: Prediction of absolute solvation free energies using molecular dynamics free energy perturbation and the OPLS force field. J Chem Theory Comput. 2010; 6(5): 1509â1519. PubMed Abstract | Publisher Full Text\n\nNaresh P, Selvaraj A, Shyam Sundar P, et al.: Targeting a conserved pocket (n-octyl-β-D-glucoside) on the dengue virus envelope protein by small bioactive molecule inhibitors. J Biomol Struct Dyn. 2020; 40(11): 4866â4878. PubMed Abstract | Publisher Full Text\n\nBIOVIA: Discovery studio visualizer. v21.1.0.20298. Dassault SystÚmes; San Diego, CA, USA: Dassault SystÚmes, 2021.\n\nSehnal D, Bittrich S, Deshpande M, et al.: Mol* Viewer: Modern web app for 3D visualization and analysis of large biomolecular structures. Nucleic Acids Res. 2021; 49(W1): W431âW437. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDaina A, Michielin O, Zoete V: SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep. 2017; 7: 42717. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPires DE, Blundell TL, Ascher DB: pkCSM: Predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures. J Med Chem. 2015; 58(9): 4066â4072. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSama-ae I: In vitro RAW DATA.xlsx.figshare. [Dataset]. 2022. http://www.doi.org/10.6084/m9.figshare.21213563\n\nSama-ae I: Raw data for the prediction of the three-dimensional structures.figshare. [Dataset]. 2022. http://www.doi.org/10.6084/m9.figshare.21214184\n\nSama-ae I: F1000_raw figures.figshare. [Dataset]. 2022. http://www.doi.org/10.6084/m9.figshare.21312297\n\nSama-ae I: Raw data for Molecular docking.figshare. [Dataset]. 2022. http://www.doi.org/10.6084/m9.figshare.21214079\n\nSama-ae I: Raw data for Molecular dynamics (MD) simulation.figshare. [Dataset]. 2022. http://www.doi.org/10.6084/m9.figshare.21214160\n\nMazal H, Aviram H, Riven I, et al.: Effect of ligand binding on a protein with a complex folding landscape. Phys Chem Chem Phys. 2018; 20(5): 3054â3062. PubMed Abstract | Publisher Full Text\n\nSama-ae I: MD simulations Movie.rar.figshare. [Dataset]. 2022. http://www.doi.org/10.6084/m9.figshare.21213560\n\nLipinski CA: Lead- and drug-like compounds: The rule-of-five revolution. Drug Discov Today Technol. 2004; 1(4): 337â341. PubMed Abstract | Publisher Full Text\n\nGhose AK, Viswanadhan VN, Wendoloski JJ: A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases. J Comb Chem. 1999; 1(1): 55â68. PubMed Abstract | Publisher Full Text\n\nVeber DF, Johnson SR, Cheng HY, et al.: Molecular properties that influence the oral bioavailability of drug candidates. J Med Chem. 2002; 45(12): 2615â2623. PubMed Abstract | Publisher Full Text\n\nEgan WJ, Merz KM Jr, Baldwin JJ: Prediction of drug absorption using multivariate statistics. J Med Chem. 2000; 43(21): 3867â3877. PubMed Abstract | Publisher Full Text\n\nMueggeI I, Heald SL, Brittelli D: Simple selection criteria for drug-like chemical matter. J Med Chem. 2001; 44(12): 1841â1846. PubMed Abstract | Publisher Full Text\n\nToretiVC, Sato HH, Pastore GM, et al.: Recent progress of Propolis for its biological and chemical compositions and its botanical origin. Evid Based Complement Alternat Med. 2013; 2013: 697390. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSforcin JM: Biological properties and therapeutic applications of Propolis. Phytother Res. 2016; 30(6): 894â905. PubMed Abstract | Publisher Full Text\n\nAsfaram S, Fakhar M, Keighobadi M, et al.: Promising anti-protozoan activities of Propolis (bee glue) as natural product: A review. Acta Parasitol. 2021; 66(1): 1â12. PubMed Abstract | Publisher Full Text\n\nTopalkara A, Vural A, Polat Z, et al.: In vitro amoebicidal activity of Propolis on Acanthamoeba castellanii. J Ocul Pharmacol Ther. 2007; 23(1): 40â5. PubMed Abstract | Publisher Full Text\n\nKuropatnicki AK, Szliszka E, Krol W: Historical aspects of propolis research in modern times. Evid Based Complement Alternat Med. 2013; 2013: 964149. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDantas Silva RP, Machado BA, Barreto GA, et al.: Antioxidant, antimicrobial, antiparasitic, and cytotoxic properties of various Brazilian Propolis extracts. PLoS One. 2017; 12(3): e0172585. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurek-Górecka A, Keskin Å, Bobis O, et al.: Comparison of the antioxidant activity of Propolis samples from different geographical regions. Plants (Basel). 2022; 11(9): 1203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVural A, Polat ZA, Topalkara A, et al.: The effect of Propolis in experimental Acanthamoeba keratitis. Clin Exp Ophthalmol. 2007; 35(8): 749â54. PubMed Abstract | Publisher Full Text\n\nBurdock GA: Review of the biological properties and toxicity of bee Propolis (Propolis). Food Chem Toxicol. 1998; 36(4): 347â63. PubMed Abstract | Publisher Full Text\n\nBlanton WE, Villemez CL: Molecular size and chain length distribution in Acanthamoeba cellulose. J Protozol. 1978; 25(2): 264â267. Publisher Full Text\n\nHuang FC, Shih MH, Chang KF, et al.: Characterizing clinical isolates of Acanthamoeba castellanii with high resistance to polyhexamethylene biguanide in Taiwan. J Microbiol Immunol Infect. 2017; 50(5): 570â577. PubMed Abstract | Publisher Full Text\n\nMoore MB, McCulley JP, Newton C, et al.: Acanthamoeba keratitis. a growing problem in soft and hard contact lens wearers. Ophthalmology. 1987; 94(12): 1654â61. PubMed Abstract | Publisher Full Text\n\nLeitsch D, Köhsler M, Marchetti-Deschmann M, et al.: Major role for cysteine proteases during the early phase of Acanthamoeba castellanii encystment. Eukaryot Cell. 2010; 9(4): 611â8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAqeel Y, Iqbal J, Siddiqui R, et al.: Anti-acanthamoebic properties of resveratrol and demethoxycurcumin. Exp Parasitol. 2012; 132(4): 519â23. PubMed Abstract | Publisher Full Text\n\nMahboob T, Azlan AM, Tan TC, et al.: Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent chlorogenic acid in vitro. Asian Pac J Trop Med. 2016; 9(9): 866â871. PubMed Abstract | Publisher Full Text\n\nLakhundi S, Khan NA, Siddiqui R: The effect of environmental and physiological conditions on excystation of Acanthamoeba castellanii belonging to the T4 genotype. Parasitol Res. 2014; 113(8): 2809â2816. PubMed Abstract | Publisher Full Text\n\nChávez-MunguÃa B, Omaña-Molina M, González-Lázaro M, et al.: Ultrastructural study of encystation and excystation in Acanthamoeba castellanii. J Eukaryot Microbiol. 2005; 52(2): 153â158. PubMed Abstract | Publisher Full Text\n\nZulhendri F, Chandrasekaran K, Kowacz M, et al.: Antiviral, antibacterial, antifungal, and antiparasitic properties of Propolis: A review. Foods. 2021; 10(6): 1360. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeal RA: Enzymic proteolysis by Entamoeba histolytica; biochemical characteristics and relationship with invasiveness. Parasitology. 1960; 50: 531â550. PubMed Abstract | Publisher Full Text\n\nPanjwani N: Pathogenesis of Acanthamoeba keratitis. Ocul Surf. 2010; 8(2): 70â79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMitsuwan W, Sangkanu S, Romyasamit C, et al.: Curcuma longa rhizome extract and Curcumin reduce the adhesion of Acanthamoeba triangularis trophozoites and cysts in polystyrene plastic surface and contact lens. Int J Parasitol Drugs Drug Resist. 2020; 14: 218â229. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMitsuwan W, Sin C, Keo S, et al.: Potential anti-Acanthamoeba and anti-adhesion activities of Annona muricata and Combretum trifoliatum extracts and their synergistic effects in combination with chlorhexidine against Acanthamoeba triangularis trophozoites and cysts. Heliyon. 2021; 7(5): e06976. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoo SY, Aung JM, Shin M, et al.: The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba. Parasit Vectors. 2020; 13(1): 368. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarate M, Marchant J, Cubillos I, et al.: In vitro pathogenicity of Acanthamoeba is associated with the expression of the mannose-binding protein. Invest Ophthalmol Vis Sci. 2006; 47(3): 1056â62. PubMed Abstract | Publisher Full Text\n\nAqeel Y, Siddiqui R, Manan Z, et al.: The role of G protein coupled receptor-mediated signaling in the biological properties of Acanthamoeba castellanii of the T4 genotype. Microb Pathog. 2015; 81: 22â7. PubMed Abstract | Publisher Full Text\n\nReliga AA, Waters AP: Sirtuins of parasitic protozoa: In search of function(s). Mol Biochem Parasitol. 2012; 185(2): 71â88. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "157123",
"date": "03 Jan 2023",
"name": "Akm Moyeenul Huq",
"expertise": [
"Reviewer Expertise Natural product Pharmacology",
"Computer aided drug discovery",
"Herbal medicine and phytherapy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work focused on the potential of Propolis extract obtained from 3 different places of Iran against Acanthamoeba. The activity was screened by MIC calculation and the potentiality of the extract as Acanthamoeba inhibitor were accessed in two different phases of life cycle. Phytochemical analysis was done by GC-MS and molecular docking followed by MD simulation were done to see the interaction of phytochemicals with target proteins. The manuscript is well organized and well written. It has the merit to be published in F1000Research journal. However, the following comments should be addressed before indexing:\nIn the MIC determination, chlorohexidine was used as positive control which is a standard drug, but why PMSF was used for anti-ensystation and anti-excystation assay and not chlorohexidine? Does the author want to relate with protease inhibition mechanism of Propolis? If so, then why the author choose different protein targets for docking and MD studies? There is lack of consistency between in vitro and in silico studies.\n\nThere is no explanation in the discussion section why Chrysin bound to a different binding site on AcMBP. Which binding site is the active site?\n\nThere is no standard value for the Pharmacokinetics and toxicity study. Make a footnote under the Table 5.\n\nLigands RMSD and RMSF are not small. The ligand RMSD stays in the trajectory does not necessarily mean the ligand-receptor complex is stable. It can move around the starting pose. The authors should provide a structure overlay comparing the starting pose and a representative snapshot from the MD.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9288",
"date": "07 Feb 2023",
"name": "Suthinee Sangkanu",
"role": "Author Response",
"response": "Thank you for all comments. Below are our point by point reply for your further consideration. Question 1: In the MIC determination, chlorohexidine was used as positive control which is a standard drug, but why PMSF was used for anti-encystation and anti-excystation assay and not chlorohexidine? Answer: Chlorhexidine is a protein synthesis inhibitor that is positively charged and interacts with the parasiteÂŽs negatively charged plasma membrane, resulting in the membrane structure that gives rise to permeability modulation, ionic leakage and cytoplasmic disruptions causing cellular damage and cell death. This drug inhibits cell growth but it has no observable effect on the encystment (Leitsch et al., 2010). Question 2: Does the author want to relate with protease inhibition mechanism of Propolis? If so, then why the author chooses different protein targets for docking and MD studies? There is lack of consistency between in vitro and in silico studies. Answer: From our study Propolis extract is suitable for inhibiting the encystment process like a positive control (PMSF). However, the main mechanism underlying inhibition of encystation by Propolis remain largely unknown. Encystation-related various proteases have been shown to be upregulated and to play an important role in cyst formation. However, how the expression of encystation-related factors is controlled during the encystation process remains unclear. In molecular docking study, sirtuins have been chosen because they are positively associated with a longer life span in some organisms and the potential regulation of pathways mediated by nutrient starvation. They have also been connected to numerous human diseases, thus sirtuin inhibitors have attracted significant attention as potential therapeutics (Joo et al., 2020). Question 3: There is no explanation in the discussion section why Chrysin bound to a different binding site on AcMBP. Which binding site is the active site? Answer: We added the explanation that why Chrysin is bound to a different binding site on AcMBP in the discussion section following the reviewer's suggestion. See line 782-792 âAfter blind docking of the three ligands with the AcMBP, the Chrysin bounded to a different binding site on AcMBP. AcMBP is a virulence factor of the free-living amoeba, which is important for adhesion of the pathogen 72. We hypothesize that this protein might have more than one binding site to help them adhere to the surface. To test this hypothesis, we predicted all this protein's binding sites with PrankWeb 74. The results are consistent with the hypothesis. The results showed that this protein has more than one pocket and some pockets have similar probability scores (Supplementary Figure 4 and Supplementary Table 1-2 43). It might be possible that this protein has more than one binding site.â Question 4: There is no standard value for the Pharmacokinetics and toxicity study. Make a footnote under the Table 5. Answer: We added the standard value for the pharmacokinetics and toxicity as a footnote under Table 5 following the reviewer's suggestion. See line 664-649 âStandard value for the Pharmacokinetics as follow: Caco2 permeability >0.90 = High [High = Good]. Intestinal absorption (human) >30% = Good. Skin Permeability > -2.5 = low [Low = Not Good]. Log VDss < -0.15 = low [Low = Good]. Log VDss > 0.45 = high [High = Not Good]. logBB > 0.3 = readily cross the BBB. logBB < -1 = poorly distributed to the brain. logPS > -2 = penetrate the CNS. logPS < -3 = unable to penetrate the CNS. Minnow toxicity (log LC50) < -0.3 = high acute toxicity 36.â Question 5: Ligands RMSD and RMSF are not small. The ligand RMSD stays in the trajectory does not necessarily mean the ligand-receptor complex is stable. It can move around the starting pose. The authors should provide a structure overlay comparing the starting pose and a representative snapshot from the MD. Answer: We added the picture of the structure compared between the starting pose and every 20 ns (200 frames) snapshot from the MD as Supplementary Figures 1-343."
}
]
},
{
"id": "157400",
"date": "03 Jan 2023",
"name": "Ascel Samba-Louaka",
"expertise": [
"Reviewer Expertise Cell biology of Acanthamoeba encystment"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this research article, Imran Sama-ae and collaborators highlight the importance of using propolis extracts as inhibitors of Acanthamoeba pathogenicity (encystment, excystment, and adhesion). They have found 3 compounds (pinocembrin, chrysin, and tectochrysin) of propolis extracts that could inhibit Acanthamoeba encystment. Using different approaches in structural biology, they suggest that the 3 compounds can bind the Acanthamoeba castellanii Sir2 protein (AcSir2) and may be excellent candidates for the development of drugs against Acanthamoeba.\nMy main comment is about the use of DMSO as a negative control. This control is missing in figures 1 and 2 and table 2. Such control is important as some studies reported DMSO as an inducer of encystment (Siddiqui et al., 20161 and Tania Martin-Pérez et al., 20212). We also need this information to understand why the % of encystment is correlated to the concentration of propolis extract (we could expect to have fewer cysts with the highest concentration). The authors have to discuss this point.\nMinor:\nIn which solution the PMSF was solubilized?\n\nIf possible, it could be nice to evaluate the percentage of pinocembrin, chrysin, and tectochrysin in the two other samples of propolis (from Sardasht county and Boroujen city).\n\nI am not a specialist, so I will not comment on the structural biology aspects. However, can the authors explain why they selected only AcSir2, AcMBP and AcGPCR as host substrates?\n\nUnless I missed it, the authors should provide the list of the 52 compounds identified in the propolis extract.\n\nPinocembrin, chrysin, and tectochrysin seem to bind and inhibit CYP1A2, CYP2C19 and CYP2C9. Isn't this an obstacle to their use as a drug in humans?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9289",
"date": "07 Feb 2023",
"name": "Suthinee Sangkanu",
"role": "Author Response",
"response": "Question 1: My main comment is about the use of DMSO as a negative control. (1) This control is missing in Figures 1 and 2 and Table 2. (2) Such control is important as some studies reported DMSO as an inducer of encystment (Siddiqui et al., 2016 and Tania Martin-Pérez et al., 2012). (3) We also need this information to understand why the % of encystment is correlated to the concentration of Propolis extract (we could expect to have fewer cysts with the highest concentration). The authors have to discuss this point. Answer: (1) In this study, we diluted the Propolis extract from stock solution (100 mg/mL) that dissolved with 99% DMSO to 1 mg/mL. This dilution made the percentage of DMSO is lower than 1%. So, in untreated condition in all experiments, we used 1% DMSO as a negative control. (2) Tania Martin-Pérez et al. (2012) reported the effect of DMSO in A. polyphaga 2961 and A. griffini MYP2004 encystation. 1.25% DMSO induced the encystation but not effect at 0.625%. Siddiqui et al. (2016) showed that 3% DMSO induced amoebae encystation in A. castellanii ATCC 50492. In our negative control (1% DMSO), the results showed that 1% DMSO not effect on encystation in both strains of A. castellanii. (3) In this study Propolis extract at low concentration (1/16 MIC) was able to inhibit the encystation of A. castellanii. The low concentration caused a reduction in the level of encystation of around 80-90%. Example from ATCC30010 showed immature cyst in low concentration (3.9 µg/mL), these immature cysts were destroyed by 0.5% SDS in the counting method. While the high concentrations (31.25 µg/mL) gave a <20% reduction in the encystment levels. And at MIC concentration (62.5 µg/mL), Acanthamoeba cells were removed by Propolis extracts. Adaptation to various adverse conditions of Acanthamoeba is an essential for the survival under unfavorable conditions. We therefore suggest that low concentration of Propolis extracts is suitable for inhibiting the encystment process. Question: In which solution the PMSF was solubilized? Answer: PMSF can dissolve in DMSO (Reference: Song F, Han X, Zeng T, Zhang C, Zou C, Xie K. Changes in beclin-1 and micro-calpain expression in tri-ortho-cresyl phosphate-induced delayed neuropathy. Toxicol Lett. 2012;210(3):276-84). Question: If possible, it could be nice to evaluate the percentage of pinocembrin, chrysin, and tectochrysin in the two other samples of Propolis (from Sardasht county and Boroujen city). Answer: Thank you for the suggestion. We are sorry to inform you that we are unable to continue this project due mainly to insufficient funds at the present. Nevertheless, we will try to continue another 2 samples of Propolis if we will be able to secure more funds in the near future. Question: I am not a specialist, so I will not comment on the structural biology aspects. However, can the authors explain why they selected only AcSir2, AcMBP and AcGPCR as host substrates? Answer: AcSir2 was classified as a class-IV sirtuin. This protein exhibited functional SIRT deacetylase activity, localized mainly in the nucleus, and its transcription was upregulated during encystation. Acanthamoeba mannose-binding protein (AcMBP) is a virulence factor of the free-living amoeba, which is important for adhesion of the pathogen. G proteins and GPCRs are well known key regulators of cellular communication and cellular functions including cell cycle, mitosis, and proliferation. Question: Unless I missed it, the authors should provide the list of the 52 compounds identified in the Propolis extracts. Answer: Supplementary Table 143. Chemical compositions of the Propolis extract sampled in Kermanshah city present in Supplementary43. Question: Pinocembrin, chrysin, and tectochrysin seem to bind and inhibit CYP1A2, CYP2C19 and CYP2C9. Isn't this an obstacle to their use as a drug in humans? Answer: We also added this critical point in the discussion section. See line 807-814 âHowever, pinocembrin, chrysin, and tectochrysin seem to bind and inhibit Cytochrome P450, including CYP1A2, CYP2C19 and CYP2C9. Because Cytochrome P450, which is primarily found in the liver, is a crucial detoxification enzyme in the body. This enzyme oxidises xenobiotics to help their excretion. In addition, the cytochrome P450 system can activate and deactivate many drugs36. Therefore, these agents should be used carefully in patients taking other drugs to avoid drug-drug interaction.â A further step that we are contemplating for future work is to work with derivatives of these three compounds and see whether they inhibit the cytochromes or not, while at the same time prove suitable as potential drugs."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1274
|
https://f1000research.com/articles/11-711/v1
|
28 Jun 22
|
{
"type": "Software Tool Article",
"title": "DEVEA: an interactive shiny application for Differential Expression analysis, data Visualization and Enrichment Analysis of transcriptomics data",
"authors": [
"Miriam Riquelme-Perez",
"Fernando Perez-Sanz",
"Jean-François Deleuze",
"Carole Escartin",
"Eric Bonnet",
"SolÚne Brohard",
"Jean-François Deleuze",
"Carole Escartin"
],
"abstract": "We are at a time of considerable growth in the use and development of transcriptomics studies and subsequent in silico analysis. RNA sequencing is one of the most widely used approaches, now integrated in many studies. The processing of these data may typically require a noteworthy number of steps, statistical knowledge, and coding skills which is not accessible to all scientists. Despite the undeniable development of software applications over the years to address this concern, it is still possible to improve. Here we present DEVEA, an R shiny application tool developed to perform differential expression analysis, data visualization and enrichment pathway analysis mainly from transcriptomics data, but also from simpler gene lists with or without statistical values. Its intuitive and easy-to-manipulate interface facilitates gene expression exploration through numerous interactive figures and tables, statistical comparisons of expression profile levels between groups and further meta-analysis such as enrichment analysis, without bioinformatics expertise. DEVEA performs a thorough analysis from multiple and flexible input data representing distinct analysis stages. From them, it produces dynamic graphs and tables, to explore the expression levels and statistical differential expression analysis results. Moreover, it generates a comprehensive pathway analysis to extend biological insights. Finally, a complete and customizable HTML report can be extracted for further result exploration outside the application. DEVEA is accessible at https://shiny.imib.es/devea/ and the source code is available on our GitHub repository https://github.com/MiriamRiquelmeP/DEVEA.",
"keywords": [
"Bioinformatics",
"transcriptomics",
"RNA sequencing",
"differential expression analysis",
"enrichment analysis",
"visualization",
"R",
"Shiny",
"interactive reports."
],
"content": "Introduction\n\nRNA sequencing (RNA-seq) has become a routine and popular technique for genome-wide and transcriptomics expression analysis.1 As a result, RNA based measurements and data are extensively incorporated in basic science research and are even increasingly used as molecular diagnostics for human health. These may include diagnosis, prognosis and therapeutic selection.2\n\nHowever, in order to leverage the full power of this technique, several stages and tools are necessary to translate expression profiles into valuable outcomes. The R statistical environment3 provides many well-known packages to perform key steps of a complete RNA-seq analyses pipeline. Possible examples include differential expression analysis (DEA) functions, leading to lists of differentially expressed genes (DEGs), and annotation enrichment analysis (EA, sometimes called pathway analysis) libraries, which will identify biological pathways or cellular functions significantly enriched from the list of DEGs. Nevertheless, most of these powerful packages are command-line based or demand coding knowledge and are therefore out of reach for scientists with limited computational training. Besides, analyses can be started at different points in the workflow, from raw or partially analysed data from different tools, to individual lists of favourite final features. This is often a limiting factor for the use of rigid tools that import only a single data type. Hence, providing flexible user-friendly tools for the analysis and visualization of gene expression data can help researchers to convert high-throughput genomics into basic science research insights. To bridge this gap, an increasing number of software tools are being released, based on intuitive, point-and-click, graphical interfaces. Frameworks such as R Shiny,4 an R package, facilitate the creation and release of interactive web tools. Some RNA-seq analysis applications from the literature may include iDEP,5 GENAVi6 and ideal,7 among others.\n\nDespite the great progress to facilitate transcriptomics computational analyses, some improvements are still possible for these tools. To this end, we have developed DEVEA, a new interactive R Shiny application for differential expression analysis, data exploration, data visualization and functional enrichment analysis. DEVEA provides an easy-to-use interface to load data in various formats and complexity according to the stage of the analysis (including raw RNA-seq count data, pre-analyzed data, simple lists of genes or proteins obtained from any source or technique, with or without statistical values associated). From them, it generates a wide set of dynamic plots and tables allowing quick navigation through the gene expression profile or enrichment analysis results. The outputs can be downloaded easily and the user can also create custom and operable reports in HTML format. DEVEA is implemented as a publicly available web server and can also be downloaded to be used locally. DEVEA aims to conduct a proper analysis by reaching out both life scientists (gathering the biological expertise) and bioinformaticians, and to foster communication between the two sides to promote easier and more extensive analysis of data.\n\n\nMethods\n\nDEVEA was built as a Shiny application4 in R3 (V.4.1.1). Shiny is a package that facilitates the development of web application from R. It is particularly indicated for building interactive and user-friendly software wrapper.\n\nThe tool is hosted on a remote web server (http://shiny.imib.es/devea), freely accessible. Apart from DEVEAâs public web server, the application can be used on a local computer (see the supplementary material for a detailed procedure as extended data here https://github.com/MiriamRiquelmeP/DEVEA/blob/main/Supplemental-Information.txt). Its source code is available on GitHub (https://github.com/MiriamRiquelmeP/DEVEA), under the terms of the Apache license 2.0. DEVEA has been tested in Linux and Windows 10 operating systems locally, and has also been launched remotely with different browsers (Google Chrome, Mozilla Firefox and Internet Explorer). Locally running the application shares all of the same characteristics as the Shiny web application.\n\nDEVEA relies on several existing R packages to carry out all the functionalities proposed (see supplementary material for the complete list). For instance, in order to handle the calculation of differentially expressed genes, the analysis is largely based on DESeq2 package.8 The annotation is managed by the AnnotationDbi package,9 collecting all the dedicated annotation databases for the different species (currently Homo sapiens, Mus musculus and Rattus norvegicus) for robust name conversion. For the enrichment calculation and visuals, the R packages topGO,10 fgsea (Fast Gene Set Enrichment Analysis)11 and clusterProfiler12 together with other basic dependencies, such as ggplot213 and plotly14 has been used.\n\nThe full DEVEA global workflow is shown on Figure 1. The main analysis path can be launched at different steps depending on the 4 input modes (check Data requirement section for details), represented at the top of the figure. The dashed arrows indicate where every input starts being incorporated in the flow, until the end of the workflow. More complex objects will generate more results. For each step of the analysis, intermediate results are available as tables and graphical representations in their dedicated spaces, detailed bellow in the circular notes. The vast majority of tables and plots are interactive, allowing the user to visualize data in real-time as well as to interact efficiently and can be individually downloaded. In the end, a global report can be generated and annotated. Each of these steps of the complete analysis will be described in their corresponding sections in the next paragraphs.\n\nThe tool has four main data input modes (Figure 2):\n\n1. CM + SI mode: refers to a counting matrix (CM), containing the raw number of counts per gene as round digits, where columns correspond to samples and rows to features. Data from other sources containing decimals must be rounded before being included in the tool. The CM should be associated with another file gathering the sample information (SI), as a data frame comprising metadata about each sample, being the first column the identifier used by default as a label in the visualizations. This can be modified afterwards during the analysis. It should include any other relevant experimental factor (e.g. treatment/control, sex, cell type, tissue, etc). The design of the comparison will be determined by one of these factors. The column names in the CM and the first row names in SI must be equal, and the gene IDs in this file can be included in Symbol or ENSEMBL format. Both files can be in .CSV, .TXT or .XLSX format.\n\n2. DO mode: based on a DeseqDataSet object (DO) generated by the DESeq() function from DESeq2 package. It is an object used to store the input values, intermediate calculations and results from a differential expression analysis. The user must have created it with the CountData field as the data matrix of counts, the ColData field with the sample information and a design formula specifying the experimental level to test for DEA. The first column in the CountData and the first row in the ColData are equal. The gene names can be included as gene Symbols or ENSEMBL gene IDs. The object must be compressed and extracted from R in .RDS format. If the differential expression object has been generated with a different tool or package, you may use the DEFormats15 R function for a possible conversion.\n\n3. GL + SV mode: a gene list (GL) with associated statistical values (SV) per gene. The first column should contain gene names (in Symbol or ENSEMBL format), the second column the fold-change and the third column the statistical adjusted p-value, in this precise order. Column names should be provided without special characters (i.e. GeneName, FC, padj). The values will be used without further modifications by DEVEA to set the threshold of expression change and the significance. This table can be uploaded in .CSV, .TXT or .XLSX file format.\n\n4. GL mode: a Gene list (GL) consisting on a single column file in .CSV, .TXT or .XLSX format containing the gene names (in Symbol or ENSEMBL) and including a column name without special characters (i.e. GeneName, Genes, ID, etc). The gene list can be copied and pasted directly into then the dedicated field in the application, without the column name.\n\nâFeatureâ represents a Gene Symbol or ENSEMBL gene ID.\n\nWhile the main data type for DEVEAâs usage is RNA-seq data, it is worth noticing that other types of âomicsâ can be used as input for the different modes. Obviously, the simple gene list (GL) can be build from any type of experiment, as long as the identifiers are recognized by the system. The more elaborated input data types, such as the counting matrix (CM + SI), can be built in some cases from different data where they can safely be processed by DESeq2 package. An example may be mass spectrometry data, that has become the method of choice for quantitative proteomics and can now assess protein samples with a respectable throughput. With label-free proteomics, it is possible to quantify proteins by using their spectral counts as an approximation of protein abundance, and then use statistical models such as DESeq2 although they are designed specifically for count data. In a study comparing different statistical methods for differential expression detection in label-free mass spectrometry proteomics, it was shown that DESeq2 performed well both in terms of detection of true positives as well as controlling for the number of false negatives.16 Therefore it is perfectly possible to use this type of data in DEVEA, as long as the values represent unique measurements as integer numbers, and replacing the protein IDs by their coding gene name.\n\nAnother example could be the use of GL + SV mode with treated data from microarray analysis, where values such as FC of expression between groups and adjusted p-value are available from the signal intensity of the probes. It is highly recommended to work with log2FC and adjusted p-values.\n\nGetting started\n\nTo start working with DEVEA, the adequate module to perform the analysis has to be chosen from DEVEAâs main lobby interface. The decision depends on the input data format. The user has to choose âGo DESeq DEVEAâ if their input is a CM + SI or a DO, whereas the âGo Simple DEVEAâ mode must be selected in the case of a GL + SV or a simple GL input files. See Figure 3 for a visual screenshot of the lobby.\n\nData upload and statistical design specification\n\nWithin the appropriate interface according to the data type, the first tab available corresponds to the âInput dataâ section. The user has to upload their own data in one of the different accepted formats and types (see them on Data requirement section) in their dedicated spaces (see Figure 4A). For all input data, a field to specify the custom dataset to use as a background universe is available as well (see Figure 4B). If necessary for the user, some demo data representing the 4 different input types, can be found on GitHub https://github.com/MiriamRiquelmeP/DEVEA/Data. The nature of the demo data and how it was generated is described in the Use case section.\n\nA: upload spaces for different types of input data. B: section to specify a custom background universe for EA.\n\nWhen a CM + SI or a DO are used as input data, it is important to indicate the statistical design or contrast for the expected comparison. The design formula expresses the variables which will be used in modeling to calculate the differential expression in following steps. For the CM + SI input format, the levels of interest that will compose the final design must be included in one of the columns of SI file. By entering the column name in the âSelect column for contrastâ field, the program extract the conditions and calculates the combination based on the distinct levels (i.e. Treatment_Control_vs_Treatment if column Treatment is selected or Sex_Male_vs_Female will be displayed if Sex is selected from SI in Figure 2 - CM + SI). In cases where more than 2 levels are available, the application will propose all 1 vs 1 combinations of them. Then, the most relevant combination for the analysis has to be selected by the user in the âSelect designâ part.\n\nWith a DO data type, the column for the design must have been already incorporated when generating the DESeq2 object in R. Only simple designs will be generated and/or can be selected from âSelect designâ field (i.e. Sex_Male_vs_Female if design = Sex is specified in the formula as in Figure 2 - DO).\n\nDifferential expression analysis (DEA) and data view\n\nThe first key performance of the application consists in extracting the descriptive information based on the feature expression and the statistical contrast representing the differential expression analysis part. In CM + SI data type a new DeseqDataSet object is calculated from the files and information provided by the user. In DO or GL + SI input modes, the application retrieves the important values already included in the objects. All transformations, normalization and measurements applied to the data at this step are performed with functions included in DESeq2 R package. It should be noted that DEA calculation is not possible with the simple GL input mode, due to the lack of expression values and statistical details. Following comments will not apply to this object.\n\nThe calculations and the statistical results are accessible in the âPreview datasetâ section tab. The user can explore at this step of the analysis the number of features considered as differentially expressed and their direction, and establish the descriptive statistics thresholds to consider them DEGs. By default, DEVEA uses prefixed log fold change|lfc|> 0.5 and p-value < 0.05 thresholds, that can be adapted by the user at any moment and thus modulate the list of DEGs. Moreover, the information uploaded and the descriptive statistics will be used to establish and control some interactive parts of the plots. For example, the color of defined up-regulated or down-regulated genes can be chosen (Figure 5A). For the first two input objects (CM + SI and DO), a complete table of results, named âStatistical - Expression valuesâ, is displayed showing useful information such as base means across samples, log2 fold changes, standard errors, raw and adjusted p-values for the specific design selected. A second table is also shown with the DEA analysis details called âSamples information - Coldataâ (Figure 5B). For the GL + SV mode, raw data are displayed in a table. The user can monitor gene name conversion, explore values interactively and sort, filter and download them at any time.\n\nA: controls used to set the statistical thresholds and cosmetics parameters. B: interactive result table.\n\nIt is important to stress that, as different elements can be extracted from the distinct input objects depending on their complexity, the number of graphs available for each of them will vary (Figures 1 and 6). Using the raw expression values that are available only in CM + SI and DO input modes, the user can explore data in a Principal Component Analysis plot (PCA) with the top 500 variant features, to show clusters of samples based on their similarity selecting the principal components of interest; a box or violin plot for gene expression distribution across the dataset; a heatmap representation of the top variant genes, regulated by the user; and a dot plot with the expression of the top 6 variant genes or a selection of individual genes of interest (Figure 7). A second group of graphs represents the plots that allow to visualize genes related only to their statistical values. This can be displayed from CM + SI, DO and GL + SV modes. They consist in a volcano plot, that shows statistical significance (adjusted p-value) versus the magnitude of change (FC) regarding the contrast levels; and a karyotype plot showing the DEG position on the genome and the direction of change (color coded by up- or down-regulated) (Figure 8B). Finally, it is also possible to combine gene expression with statistical values, from CM + SI and DO input modes, to generate a MA plot (an application of a BlandâAltman plot for visual representation of genomics data17) (Figure 8A) displaying feature labels and statistical values by clicking on each gene dot.\n\nA: PCA plot. B: box and violin plots. C: gene expression heatmap. D: sample hierarchical clustering. E: dot plots.\n\nA: section to explore volcano and MA plots. B: karyotype plot.\n\nEnrichment analysis (EA) and visualization\n\nThe last stage of the analysis with DEVEA is EA. This is a method to identify classes of defined categories that are over-represented in the list of DEGs. These categories may be associated with disease phenotypes, biological pathways, or cellular functions. DEVEA uses the differentially expressed and significant features to retrieve the over-represented terms from several well known databases. This major block of the DEVEA analysis can be carried out from all data input types. It consists of an extensive EA after the selection of appropriate statistical values for defining DEGs from CM + SI, DO and GL + SV, or using all components included in the simple GL input. It collects significant terms from KEGG (Kyoto Encyclopedia of Genes and Genomes18) and GO (Gene Ontology19) Biological process, Molecular function and Cellular component databases. Furthermore, a GSEA-type analysis (Gene Set Enrichment Analysis20) can be performed from different databases for the whole set of features. In the case of CM + SI, DO and GL + SV input modes, KEGG and GO analyses are performed for all DEGs together, and for the subset of up- and down-regulated genes in separated tabs. GSEA analysis is always performed on the complete set of genes and uses the statistical values associated with the features. With the simple GL data input, enrichment can only be performed for the whole set of genes for KEGG and GO analysis and no GSEA analysis will be possible, due to the lack of statistical information.\n\nIn the EA performed by DEVEA, the main results are shown as interactive tables containing detailed information on the enrichment from each database. In KEGG and GO categories, the tables display columns for the name of the significant pathways or terms, their (adjusted) p-values and additional descriptive information such as total number of genes associated or the DEGs participating in the pathway. The user can also display the feature names that match in the pathway from the + symbol. Below each table, additional plots can be created by selecting rows of interest (showed in green on the Figure 9A). The plots are interactive and reactive. They can be changed at any time by selecting new lines in the table. The user can visualize results as word cloud, circle plot, bars plot, chord plot, dot plot, heatmap or net plot representing different elements from the tables. For GSEA-type analysis, the results are displayed as a table containing the significant enriched pathways from the selected databases. In the table, important GSEA calculation parameters are available and below a typical GSEA plot is shown from the lines selected in the table (see Figure 9B).\n\nA: KEGG EA table and heatmap. B: GSEA-type EA table and plot.\n\nAs a special feature offered by DEVEA, a custom gene list can be loaded to be used as the background gene universe for EA (Figure 4B). For example, the user can use a background list containing only expressed genes in this experiment. It will control for experimental context and may enable the results to define not only the nature of the samples but also representative functions. This is especially important to consider in microarray studies when a limited number of probes is used, or in studies of specific cells or tissue with a restricted set of detectable genes.\n\nFor this EA, internal ENTREZ ID gene code is used to associate gene names in Symbol or ENSEMBL with the enrichment annotation in KEGG, GO and GSEA libraries. An exhaustive conversion across different functions is conducted to retrieve all possible terms, since they are not registered in all conversion databases similarly. Furthermore, not all genes are curated or annotated and therefore some will be left out of the EA (the portion of genes is indicated in the application below the âpreview tableâ as shown in Figure 5B). This is a limitation from the automated databases conversions without manual curation. For this reason, the number of species is limited and not all genes will be used for the enrichment part, but the results obtained are more robust.\n\nGlobal report\n\nAn interactive HTML report can be generated from all data types following analysis and exploration with DEVEA. It is available in the âHTML reportâ button at the top fixed part of the application. To create it, the user can select the individual set of figures, tables and results to be kept in this single html document (Figure 10A). Plots will retain the last aesthetic indicated in the graphical parameters (e.g. colours, shapes, labels, terms). Only full tables will be kept, without taking into account potential filters applied during the analysis, allowing full exploration, sorting and re-filtering of the whole data outside of the application. It should be noted that the majority of the results can also be copied or downloaded in high-quality format at any step of the analysis inside DEVEA. Importantly, comments can be included at any step of the analysis in a dedicated section at the top right position of the application, and will be automatically saved (Figure 10B). They can be displayed in the final report to ensure that the observations made throughout the analysis, with special interpretations or results are maintained.\n\nA: configuring box and final HTML report. B: notes transferable to the report.\n\nPlease note that if any of the graphs or tables do not work in the application, when there are not enough results, genes or routes to display them, the report cannot be generated with errors. Unselect the problematic plots or tables to be able to render the rest of the report.\n\n\nUse case\n\nTo demonstrate the usefulness of DEVEA, a RNA-seq dataset generated by our team and published recently21 was investigated using the application. The study aimed to characterize the role of JAK2/STAT3 signaling in astrocytes in the context of Huntingtonâs disease (HD). HD is a rare genetic neurodegenerative disease leading to severe motor, cognitive and psychiatric symptoms, with no curative treatment available.22 Astrocytes, an heterogeneous group of star-shaped glial cells perform key functions in the brain, they provide nutrients to neurons, regulate synaptic transmission, and contribute to nervous tissue repair following injury.23 Astrocytes become reactive in the brain of HD patients and their impact on HD progression is still unclear.24 The study used a genetic mouse model of HD. Treated mice were injected with an adeno-associated viral (AAV) vector targeting astrocytes and encoding a constitutive form of the JAK2 kinase (JAK2T875N) to activate the JAK2-STAT3 pathway. Control mice were injected with a similar AAV expressing GFP. Astrocytes were isolated and sequenced by RNA-seq from Illumina platform. Reads were aligned on the mouse genome (ENSEMBL GRCm38 release 96) and a count matrix was generated. Data were adapted and integrated as different input objects in DEVEA to test all functionalities. For instance, Figure 7A shows that control (GFP, N=5, in green) and treated (JAK, N=6, in red) samples are clearly separated on a PCA plot, with a better separation achieved on PC2 (representing 27% of the total variance). Figure 7C-D also show two types of clustering profile, which group samples from the same group together and display genes with higher variability. Figure 11A shows that the levels of Jak2 are higher in treated (JAK) versus control (GFP) groups, as expected by AAV-mediated gene transfer (log2(FC) = 6, associated adjusted p-value 8.7E-69). In addition, a volcano plot demonstrates that the JAK2T875N causes down-regulation of many genes DEGs, shown in blue in the upper left quartile of the graph on Figure 11B. Finally, EA in the treated (JAK) versus control (GFP) list of DEGs shows that many GO-BP terms are related to Immunity/Inflammation, a process linked to the reactive changes in astrocytes induced by JAK2 (Figure 11C).\n\nA: expression levels of the Jak2 gene between groups. B: volcano plot. C: GO-BP enriched categories.\n\nAll data corresponding to this research project are available under the four different DEVEA input types. They are available on the GitHub web site (see the Software Availability section). They consist in (1) a set of CM + SI, where features are genes in the ENSEMBL format and expression represents raw counts after alignment and filtering to remove non-expressed genes from the CM. The final number of features is 18260 + 2 custom genes representing the two transgenes JAK2T875N and GFP). The SI file contains all relevant information on sample characteristics; (2) a complete DO built from the same CM and SI files with a design based on the comparison of the two different AAVs (design = AAV); (3) a GL + SV file manually created according to the DESeq2 results. The file is generated with 3 columns that contains the gene names, FC and P-Value for the top 500 most significant genes and (4) a unique GL containing only the 268 DEGs from the comparison of interest, reported from the DESeq2 analysis with adjusted P-Value < 0.1 statistical threshold and no threshold for the FC (for further details on the input data, see the Data requirement section).\n\nWith this user case, no errors were detected throughout the analysis. DEVEA, thanks to a large range of graphics and statistical analysis highlighted significant differences between reactive astrocytes in the JAK group and control astrocytes in the GFP group. Thanks to extensive EA analysis, DEGs were associated with important biological functions such as immunity/inflammation pathways as well as cytokine signaling, proteostasis and energy metabolism. These results are consistent with the insight described earlier this year in the team.21\n\n\nRelated works (state-of-the-art)\n\nThe field of application tools for transcriptomics data visualization, DEA and EA is constantly growing. To compare DEVEA functionalities with similar applications, six recently published tools that operate through a graphical user interface were selected, provide interactive results, and are based on stable and maintained R packages. The six selected tools are iDEP,5 ShinyGO,25 DEGenR,26 GENAVi,6 RNfuzzyApp27 and ideal.7 A detailed comparison of the DEVEA main attributes is shown in Table 1. Characteristics that are related to data management and import into the application has been stressed at the beginning, followed by the various modes of DEG identification and different interactive graphical results, EA calculations and global reporting and hosting. It is clear from Table 1 that most of the selected application share many functionalities with DEVEA. One exception is the application ShinyGO, which offers significantly less options since it is designed to perform uniquely enrichment calculations from simple gene lists. However, some other specific differences exist with the rest of the tools closer in purpose to the one presented here. DEVEA has more flexibility in terms of data type import into different formats, representing different stages of the analysis. Similarly, the user has a wide range of exploration possibilities via GL and GL + SV input formats, in terms of data generation and origin. The list of features and the associated statistics may have been generated from many different external tools or could represent several analysis types as long as they are eventually converted into gene names (i.e. Microarray data results, proteomics results, gene lists from the literature, etc.). As a further advantage, the ability to import complex objects, such as DO increases the number of visualization and analysis options. Despite this, not all possible visuals that can be generated from these objects are included in the application, which thus has room for expansion. In particular, DEVEA could further develop data management functionalities, by extending the capacity of dealing with batch effect or data pre-processing and filtering. One of the possible drawbacks is the low number of available species for the EA compared with other tools or the potential mismatches when converting gene names. To counter this, numerous graphics are displayed with whom the user can interact and perform a complete EA. The EA can be generated from all DEGs, either split by the direction of expression change, or merged into a single list. The custom global report is a unique feature to DEVEA. This might be very handy to share results with collaborators, because the user can easily insert comments, and transfer the fully-interactive HTML report. Last, DEVEA appears slightly more flexible in terms of the application hosting and running, since it is possible to run it online (DEVEA web server), or offline (from R). For instance, some applications like DEGenR and RNfuzzyApp do not offer the possibility to run the application online.\n\nAlthough overall applications share common analysis blocks, DEVEA presents more graphical variety than most of them. For example, as a criteria to consider in the table that they contain âInteractive preview visualsâ to preliminary explore the data, only one of PCA plot, violin/box plot of sample profile, heatmap for gene expression, sample clustering, gene expression dots plot per groups should be generated from the applications. For âInteractive DE visualsâ of the DEA statistics and profile the criteria consist of including at least one of volcano plot, MA plot or karyoplot. Finally, for the âInteractive visualsâ to navigate the EA, the marked tools include at least one of bars plot, dots plot, chord plot, heatmap, net plot, word cloud, circle plot of the enriched terms. For most applications, only a small subset of these plots are implemented. DEVEA contains all these graphs, requirement that none of the others met.\n\n\nConclusion and future directions\n\nThe DEVEA application is developed to improve the set of existing software to perform DEA, data visualization and annotation or EA from transcriptomics data. DEVEA meets the need for applications that give sufficient usage autonomy, without compromising the complexity and accuracy of the results. It provides an interactive and user-friendly interface accessible for users without bioinformatics training, with high diversity of analysis components. Researchers can explore their data, carry statistical DEA and subsequent EA from distinct well-known databases without losing possible customization in real-time. It is a wide wrapper of functions in a single tool, avoiding the use of different tools/websites to run the distinct steps of the transcriptomics analysis and to reach this level of advanced ready-to-publish visuals, tables and results. One of the main strength is the incorporation of several input data type options. The possibility to include a custom background make DEVEA suitable for analysis in which correction of some experimental bias could lead to better results in the EA part. Another key advantage is that DEVEA let the user extract their results individually or in a still interactive way through a custom HTML format file. To further develop DEVEA analyses, we plan to later offer additional pre-treatment options (remove batch effect, filter genes by expression, etc.). More species can also be integrated. Other improvements include transcription factor enrichment analysis and use of complete datasets from different omics such as proteomics spectral counts matrix, microarray expression matrix, etc. In conclusion, a purpose of DEVEA is to promote a dialogue between biologists and bioinformaticians, particularly to produce suitable data and to understand the validity of the data needed to create the best downstream results.\n\n\nSoftware availability\n\n\n\n1. Software available at http://shiny.imib.es/devea/. Archived source code as at time of publication: https://doi.org/10.5281/zenodo.6657245.\n\n2. Latest source code on https://github.com/MiriamRiquelmeP/DEVEA.\n\n3. Test files for every input mode can be found also on https://github.com/MiriamRiquelmeP/DEVEA Data section.\n\n4. Tutorial accessible from both DEVEA modules (DESeq DEVEA and Simple DEVEA) in the âTutorialâ section from the top controls and independently on https://shiny.imib.es/DESeqDevea/tutorial.html or https://shiny.imib.es/simpleDevea/tutorial.html.\n\nLicense: Apache license 2.0.\n\n\nAuthor contributions\n\nMRP and FPS conceived the application, did the development and wrote the manuscript. CE, SB and EB supervised the work, tested the application and wrote the manuscript. JFD contributed fund acquisition and resources for the project. All authors discussed the results and contributed to the final manuscript.",
"appendix": "Acknowledgements\n\nWe would like to thank all the people from MIRCen, CNRGH and IMIB who helped in the testing for the implementation of the application and provided valuable ideas for improvement.\n\n\nReferences\n\nMortazavi A, Williams BA, McCue K, et al.: Mapping and quantifying mammalian transcriptomes by rna-seq. Nat. Methods. 2008; 5(7): 621â628. Publisher Full Text\n\nByron SA, Van Keuren-Jensen KR, Engelthaler DM, et al.: Translating rna sequencing into clinical diagnostics: opportunities and challenges. Nat. Rev. Genet. 2016; 17(5): 257â271. PubMed Abstract | Publisher Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. Vienna, Austria:R Foundation for Statistical Computing;2020.Reference Source\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. 2021. R package version 1.7.1.Reference Source\n\nGe SX, Son EW, Yao R: idep: an integrated web application for differential expression and pathway analysis of rna-seq data. BMC Bioinform. 2018; 19(1): 1â24.\n\nReyes ALP, Silva TC, Coetzee SG, et al.: Genavi: a shiny web application for gene expression normalization, analysis and visualization. BMC Genomics. 2019; 20(1): 1â9.\n\nMarini F, Linke J, Binder H: ideal: an r/bioconductor package for interactive differential expression analysis. BMC Bioinform. 2020; 21(1): 1â16.\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for rna-seq data with deseq2. Genome Biol. 2014; 15(12): 1â21. Publisher Full Text\n\nPagÚs H, Carlson M, Falcon S, et al.: AnnotationDbi: Manipulation of SQLite-based annotations in Bioconductor. 2020. R package version 1.52.0.Reference Source\n\nAlexa A, Rahnenfuhrer J: topGO: Enrichment Analysis for Gene Ontology. 2020. R package version 2.42.0.\n\nKorotkevich G, Sukhov V, Sergushichev A: Fast gene set enrichment analysis. bioRxiv. 2019. Publisher Full Text Reference Source\n\nGuangchuang Y, Wang L-G: Yanyan Han, and Qing-Yu He. clusterprofiler: an r package for comparing biological themes among gene clusters. Omics: A Journal of Integrative Biology. 2012; 16(5): 284â287.\n\nWickham H: ggplot2: Elegant Graphics for Data Analysis. New York:Springer-Verlag;2016.Reference Source\n\nSievert C: Interactive Web-Based Data Visualization with R, plotly, and shiny. Chapman and Hall/CRC;2020.Reference Source\n\nOleÅ A: Deformats: Differential gene expression data formats converter.2021. R package version 1.20.0.Reference Source\n\nLangley SR, Mayr M: Comparative analysis of statistical methods used for detecting differential expression in label-free mass spectrometry proteomics. J. Proteome. 2015; 129: 83â92. PubMed Abstract | Publisher Full Text\n\nAltman DG, Bland JM: Measurement in medicine: The analysis of method comparison studies. Journal of the Royal Statistical Society. Series D (The Statistician). 1983; 32(3): 307â317. 00390526, 14679884.Reference Source\n\nKanehisa M, Goto S: Kegg: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27â30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBotstein D, Cherry JM, Ashburner M, et al.: Gene ontology: tool for the unification of biology. Nat. Genet. 2000; 25(1): 25â29.\n\nJiang Z, Gentleman R: Extensions to gene set enrichment. Bioinformatics. 2007; 23(3): 306â313. PubMed Abstract | Publisher Full Text\n\nAbjean L, Haim LB, Riquelme-Perez M, et al.. Reactive astrocytes promote proteostasis in Huntingtonâs disease through the JAK2-STAT3 pathway. Brain. 03 2022; awac068. Publisher Full Text\n\nTabrizi SJ, Flower MD, Ross CA, et al.: Huntington disease: new insights into molecular pathogenesis and therapeutic opportunities. Nat. Rev. Neurol. 2020; 16(10): 529â546. PubMed Abstract | Publisher Full Text\n\nVerkhratsky A, Nedergaard M: Physiology of astroglia. Physiol. Rev. 2018; 98(1): 239â389. PubMed Abstract | Publisher Full Text\n\nHaim LB, Sauvage M-A C-d, Ceyzériat K, et al.: Elusive roles for reactive astrocytes in neurodegenerative diseases. Front. Cell. Neurosci. 2015; 9: 278. Publisher Full Text\n\nGe SX, Jung D, Yao R: Shinygo: a graphical gene-set enrichment tool for animals and plants. Bioinformatics. 2020; 36(8): 2628â2629. PubMed Abstract | Publisher Full Text\n\nChoudhary KS, Caldwell AB, Subramaniam S: DEGenR: An R Shiny app for differential gene expression and enrichment analysis.May 2021. Publisher Full Text\n\nHaering M, Habermann BH: Rnfuzzyapp: an r shiny rna-seq data analysis app for visualisation, differential expression analysis, time-series clustering and enrichment analysis. F1000Res. 2021; 10: 654. Publisher Full Text"
}
|
[
{
"id": "142536",
"date": "16 Aug 2022",
"name": "HélÚne Hirbec",
"expertise": [
"Reviewer Expertise Transcriptomics",
"Neuroinflammation",
"Alzheimer Disease"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present manuscript Riquelme-Perez and coll. are presenting the DEVEA software aimed at helping biologists with no or poor programming skills to analyse their Omics data (mainly RNA-seq). The rationale for the development of a new software is clearly explained, additionally in the manuscript, the authors compare their software to other web-based software and highlight that DEVEA is higher flexibility (see Table 1). The software is available as a web-based application but can also be installed locally.\nThe description of the software is technically sound. As a matter of facts, it is based on the several well-known and highly used R packages aimed at analysing RNA-seq data. Interestingly, differential expression analysis (DEA) is performed using DESeq2 package on the most reliable tool for DEA. DEVEA is also flexible as it accepts input data from different format and at different stage of the analysis. Thus, DEVEA can be used for initial analysis of the data, but can be used at later stage of the data analysis. One very interesting feature of the DEVEA software is that it enables loading a specific background universe.\nThe manuscript is clear and very well written. There is sufficient information provided in the manuscript to run the analyses proposed. However, it will be useful if the authors could provide (as supp material?) a step by step protocol for the different type of analysis (CM+SI, DO, GK+SV; GL).\nOverall, the manuscript presents a new software that appear more flexible and present several improvements compared to other similar software developed recently. DEVEA will be useful to biologists with poor programming skills to explore their omics data, in particular RNA-seq data. Although, the software is already usable, I noticed a couple of minor issues that would deserve improvements.\n\nMinor points that would need improvements:\nCould the authors specify if already normalized counts can be used as input in CM+SI type of analysis?\n\nCould the authors specify whether the software support both â.â and â,â as decimal separators;\n\nWhen uploading the GL with gene symbols, these latter are automatically converted to Ensembl identifiers, but some symbol are not recognized. It is unclear which genes are considered for EA analyses: all genes with symbol, only genes with an Ensembl identifier, only annotated genes with an Ensemble identifier?\n\nIn some of the EA graphs, deregulated pathways/gene sets are referred to by their url rather than by the pathway name. This complicates the reading of the graphs;\n\nDemo data are downloadable, but the link given in the manuscript does not work;\n\nDownloads of the SVG images donât seem to work, except for Chordplot in KEEG Enrichment for which the download is different. However, in this latter case, only the plot (but not the legend) is downloaded;\n\nThe inclusion of GSEA type results in the analysis is interesting. Including the GSEA leading edge analysis in DEVEA would be an interesting add.\n\nFurther improvements that would be interesting to make:\nEnrichment analysis: In addition to GO and KEEG analyses, it would also be interesting to add other databases, such as Wiki Pathways for example.\n\nIt will also be interesting to enable evaluating over-representation for custom genesets (i.e. specific molecular signatures of interest in the research field).\n\nRemarks: Assessment as to whether sufficient details of the code, methods and analysis is provided to allow replication of the software development and its use by others, is beyond the competence of the reviewer.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "9366",
"date": "24 Mar 2023",
"name": "Miriam Riquelme-Perez",
"role": "Author Response",
"response": "Thank you for taking the time to review our paper, and for the thoughtful suggestions. In the revised version, we have modified the main text to refer more clearly to the method information provided on the interface, and improved the clarity of the text in several places, according to your suggestions. In the same way, we have included a few enhancements in the application tool itself. Below are the questions raised by the reviewer (- in italic), followed by the authors' response (+ in bold): - The manuscript is clear and very well written. There is sufficient information provided in the manuscript to run the analyses proposed. However, it will be useful if the authors could provide (as supp material?) a step by step protocol for the different type of analysis (CM+SI, DO, GK+SV; GL). + In addition to the article and the information within DEVEA, it was also possible to consult a comprehensive walkthrough on how to use the application from the different input modes. It was accessible from both DEVEA modules (DESeq DEVEA and Simple DEVEA) in the âTutorialâ section from the top right controls inside the application interface and independently on https://shiny.imib.es/DESeqDevea/tutorial.html or https://shiny.imib.es/simpleDevea/tutorial.html. The tutorial was mentioned in the âSoftware availabilityâ section, and now this is mentioned in the âOperationâ section. - Could the authors specify if already normalized counts can be used as input in CM+SI type of analysis? + The counting matrix (CM) and the sample information (SI) elements will be used as raw data to be internally analyzed through the various functions of the DESeq2 package. To build the final DESeqDataSet object, DEVEA uses the data as obtained from the counting process, presented in the form of an array of integer values. Decimals will not be accepted. The DESeq2 model internally corrects for library size and generates its own normalization, so transformed or normalized values must not be used in the CM + SI input type (as detailed in the official bioconductor DESeq2 vignette section \"Why un-normalized counts?\" http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#why-un-normalized-counts). A sentence that specifies this point is now included in the âData requirementâ section, 1. CM + SI mode. - Could the authors specify whether the software support both â.â and â,â as decimal separators? + Thank you for raising this point. Decimals must be indicated with \".\" as a separator. \",\" will not be properly recognized by the program. - When uploading the GL with gene symbols, these latter are automatically converted to Ensembl identifiers, but some symbol are not recognized. It is unclear which genes are considered for EA analyses: all genes with symbol, only genes with an Ensembl identifier, only annotated genes with an Ensemble identifier? + The KEGG, GO, and GSEA libraries used in DEVEA need as input a list of genes coded in their ENTREZ ID format. For this reason, an exhaustive process to convert the initial gene names provided by the user (either in gene SYMBOL or ENSEMBL) into ENTREZ ID is conducted internally by DEVEA. Finally, the EA functions retrieve the associated annotation terms from these genes to generate the results. Therefore, only genes from the original data with an ENTREZ ID name can be linked to its annotation in the libraries. Unfortunately, not all genes have curated ENTREZ ID names linked with the different formats or have annotated functions associated with the libraries. As a consequence, some genes will be left out of the EA. The number of genes considered in the following steps is indicated in green below the âpreview tableâ as shown in Figure 5B. This is a limitation of the gene name conversion functions without manual curation. This point was discussed in the âEnrichment analysis (EA) and visualizationâ subsection in the âImplementationâ part. - In some of the EA graphs, deregulated pathways/gene sets are referred to by their url rather than by the pathway name. This complicates the reading of the graphs; + We agree that using only the URL is cumbersome since it is necessary to go back to the table to link with the actual name. Due to space limitations in some plots, pathways or functions that have a very long name cannot be shown in full. However, in most of the images where the full name cannot be displayed, it will be shown in full by placing the mouse arrowhead on top of the interactive graph. - Demo data are downloadable, but the link given in the manuscript does not work; + We apologize for this problem. The link has been corrected in the manuscript and now redirects to the appropriate page to access the demo data on GitHub. - Downloads of the SVG images donât seem to work, except for Chordplot in KEGG Enrichment for which the download is different. However, in this latter case, only the plot (but not the legend) is downloaded; + Indeed, there was a server issue that has now been fixed. The functionality to export SVG images is fully operational again. Some graphics may change slightly when downloaded because the functions used to render/download are not necessarily the same as those used to display them. The plot information must remain the same. - The inclusion of GSEA type results in the analysis is interesting. Including the GSEA leading edge analysis in DEVEA would be an interesting add. + We agree that the inclusion of the leading edge for GSEA results is interesting. This has been included in DEVEA as an extra column in the table at the GSEA tab, called âleading_edgeâ. It has also been implemented in the plot as a red line indicating the extent of what we consider the leading edge genes. This is also mentioned in the text âEnrichment analysis (EA) and visualizationâ section. - Further improvements that would be interesting to make : Enrichment analysis: In addition to GO and KEEG analyses, it would also be interesting to add other databases, such as Wiki Pathways for example. It will also be interesting to enable evaluating over-representation for custom genesets (i.e. specific molecular signatures of interest in the research field). + Regarding the first further improvement, adding more databases would be a significant extension to the current version of DEVEA, that we want to keep fast and operational. For the second suggestion, the specific custom genesets needed for the over-representation analysis could be very different between users and experiments, hence we prefer to only include the curated well known genesets from the databases. Nevertheless, DEVEA allows the definition of background genes for comparison in the EA part, avoiding the inappropriate inflation of statistical p-values and false-positive results from contrast against the whole genome/proteome. For the moment, we aim at offering a stable and robust version and creating a docker container with these basic functionalities efficiently working in the server. We think that including these significant improvements is beyond the scope of this first DEVEA version. However, these are exciting suggestions to be considered in the following versions. Our goal is to further develop DEVEA according to the needs of the community and bio-informatics developments. This is acknowledged in the âConclusion and Future Perspectivesâ section, and we will consider including new functionalities in a future version of DEVEA."
}
]
},
{
"id": "152235",
"date": "10 Oct 2022",
"name": "Wenbin Guo",
"expertise": [
"Reviewer Expertise Transcriptomics method development",
"automated pipeline development",
"Shiny App development",
"bioinformatics analysis",
"RNA-seq data analysis",
"differential gene expression analysis",
"differential alternative splicing analysis",
"gene regulatory network analysis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\nThe author developed a shiny App DEVEA for biologists who lack extensive bioinformatics skills to perform complex differential expression analysis and downstream function annotation and pathway enrichment analysis. It provides an easy-to-use platform and empowers biologists to hand on complex RNA-seq analysis by themselves. The multiple options of inputs, including (a) the raw read counts of genes, (b) the intermediate dataset produced by DESeq2, (c) the gene list with statistics of significance and (d) the gene list only, allowed users to start the analysis flexibly from different types of data. It also provided interactive figures and tables to visualise the results in real-time. In the end, a report can be generated with user-selected results and figures, which improved the reproducibility of the RNA-seq analysis.\nBelow are some limitations of the current DEVEA and suggestions for future improvements. The author already had a plan to address some of them in future directions.\nIt seems that the current version of DEVEA only supports the analysis of human, mouse and rat, which limits its usage in wider studies of plant species and other animal species.\n\nDue to the above limitation, the current manuscript lacks some case studies in other species, especially in plants.\n\nThe controls for errors caused by unwanted variations are not proposed in the DEVEA pipeline. For example, RNA-seq data often have batch effects between replicates and they will largely hinder the accuracy of differential expression analysis. DESeq2 also provides suggestions on how to correct the batch effects in the user manual.\n\nDEVEA only allows a pair-wise comparison between control and treatment at a time, which is not flexible for complex experimental design, such as datasets with a great number of conditions to compare, datasets of time-series data and development series, and datasets with multiple groups and each group include addition level of conditions to compare.\n\nIt would be impressive to enable enrichment analysis for the genes lacking annotations. For example, getting the gene annotation by blasting the sequence against some databases. It will be especially useful for the analysis of newly assembled genes and transcripts.\n\nComments for manuscript improvement:\nThe preparation of some input data is still difficult for biologists who lack bioinformatics skills. For example, getting the raw read counts requires bioinformatics skills to map the RNA-seq data to a reference genome or transcriptome and generate read counts by using quantification tools such as Salmon, Kallisto and RSEM. The author should propose an easy-to-access method and provide step-by-step tutorials for biologists to generate the read counts from raw RNA-seq data, for example, using the Galaxy platform.\n\nThere are massive methods available for the function of differential expression (DE) analysis. Many comparison studies showed that the methods DESeq2, Limma-voom, edgeR and Sleuth have similar performance in DE analysis and each of these methods has a big user group. To attract more users to DEVEA, the author could highlight the advantages to use DESeq2 in DEVEA compared with other methods, such as Limma and edgeR, in the introduction or discussion.\n\nIn the tutorial, the table of contents is floating and overlaps with texts and videos. Is it possible to reallocate the position of the table of contents to avoid overlapping?\n\nIn the example data link: https://github.com/MiriamRiquelmeP/DEVEA/tree/main/data, it would be better to include a readme file with descriptions of the example datasets.\n\nâSupplemental-Information.txt: Running DEVEA locally on a machineâ: I would recommend the author build DEVEA as an R package. Then, instead of âDownload the compressed folder from the DEVEA GitHub repositoryâ, users can install and run DEVEA on a local machine as an R package.\n\nPage 3: âDespite the great progress to facilitate transcriptomics computational analyses, some improvements are still possible for these tools.â Although comparisons were given in the Related work section, the author should give brief descriptions of these tools and summarise their limitations as literature reviews in the introduction. The author also should address what are the possible improvements and why they are needed.\n\nPage 11: âReads were aligned on the mouse genome (ENSEMBL GRCm38 release 96) and a count matrix was generated.â For reproducibility, the tool for read mapping and count matrix generation should be referred to and cited.\n\nFigure 7: the font size of labels and some details of the figures are too small. Would it be better to change the multiple plot layout to 3 rows x 2 columns?\nThe paper has quite a few typos. The author should carefully check the entire manuscript again. Here, I have picked out some of them:\nPage 3: âkey steps of a complete RNA-seq analyses pipelineâ should be âkey steps of a complete RNA-seq analysis pipelineâ\n\nPage 3: âdetailed bellow in the circular notesâ should be âdetailed below in the circular notesâ.\n\nPage 5: âthe simple gene list (GL) can be build fromâ should be âthe simple gene list (GL) can be built fromâ\n\nPage 6: âthe program extract the conditions and calculatesâ should be âthe program extracts the conditions and calculatesâ\n\nPage 11: âTo demonstrate the usefulness of DEVEA, a RNA-seqâ should be âTo demonstrate the usefulness of DEVEA, an RNA-seqâ\n\nPage 12: âwhich offers significantly less options sinceâ should be âwhich offers significantly fewer options sinceâ\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "9367",
"date": "24 Mar 2023",
"name": "Miriam Riquelme-Perez",
"role": "Author Response",
"response": "We thank the reviewer for taking the time to review our paper and test our application. We provide here a detailed response (in bold, after + symbol) to all the comments (introduced by -, in italic) and how we addressed the majority of them. - It seems that the current version of DEVEA only supports the analysis of human, mouse and rat, which limits its usage in wider studies of plant species and other animal species. Due to the above limitation, the current manuscript lacks some case studies in other species, especially in plants. + We acknowledge that there is limited support for plant and animal species in the current version of DEVEA. We are planning to expand the number of species available in future versions. For this version, following the reviewer's suggestion, we have now included the model plant species Arabidopsis thaliana in DEVEA. A demo dataset has also been created compatible with DEVEA for this species, based on an RNA-seq study published recently (Lee et al. New Phytologist, 2020, 225(4):1715-1731). - The controls for errors caused by unwanted variations are not proposed in the DEVEA pipeline. For example, RNA-seq data often have batch effects between replicates and they will largely hinder the accuracy of differential expression analysis. DESeq2 also provides suggestions on how to correct the batch effects in the user manual. + We can distinguish between modeling the batch effect using DESeq2 within DEVEA and removing the batch effect before importing the data into DEVEA. An advantage of using as input data a DO generated previously in R, is that the user can include Batch in the design formula as âdesign = ~Batch + Conditionâ. Once the final contrast is specified as Condition_level1_vs_level2, the batch effect will then be 'accounted for' (the statistical inferences will be adjusted for Batch) with a differential expression analysis testing for Condition. Batch is essentially treated in DEVEA as a covariate in the regression model, just as we do in association studies. If the user wants to effectively remove the batch effect for downstream analyses, it can be performed outside DEVEA on the variance-stabilized or rlog expression values using limma::removeBatchEffect() or ComBat-seq tool, before uploading the data to the application again. This could be considered for future versions of DEVEA as well. We have added a section in the âData upload and statistical design specificationâ section of the manuscript. - DEVEA only allows a pair-wise comparison between control and treatment at a time, which is not flexible for complex experimental design, such as datasets with a great number of conditions to compare, datasets of time-series data and development series, and datasets with multiple groups and each group include addition level of conditions to compare.  + Multiple experimental conditions can be included in the design of the DESeq2 object (DO). DEVEA will offer the possibility to consider any of them foreach analysis. For instance, when âdesign = ~ConditionA + ConditionB + ConditionCâ, the tool will propose as possible contrast 1.ConditionA_level1_vs_level2, 2.ConditionB_level1_vs_level2 and 3.ConditionC_level1_vs_level2. The user can select from DEVEA interface, which specific contrast to explore. In addition, for DO and CM + SI, when there are more than two levels from the same condition, DEVEA will propose all the one-vs-one combinations. Therefore, if the âdesign = ~ ConditionWith4levelsâ, the possible contrasts would be 1.Condition_level1_vs_level4, 2.Condition_level2_vs_level4, and 3.Condition_level3_vs_level4, level4 being the basal condition for every contrast. For CM + SI, these levels are selected alphabetically. In a DO, the user can use the function relevel() from DESeq2 in R to specify the basal level. We have changed the text in the manuscript to indicate these possibilities, in the âData upload and statistical design specificationâ from the âImplementationâ section. - It would be impressive to enable enrichment analysis for the genes lacking annotations. For example, getting the gene annotation by blasting the sequence against some databases. It will be especially useful for the analysis of newly assembled genes and transcripts. + We agree that such an initiative would provide a genuinely useful service, especially for recently sequenced organisms who do not have yet a good and comprehensive annotation. However, this task is far from being trivial and requires a substantial amount of software development and is therefore currently beyond the scope of the current DEVEA implementation. For DEVEA, we currently aim to exploit available packages and resources for visualization, differential expression analysis and enrichment from well-known and described species. Comments for manuscript improvement: - The preparation of some input data is still difficult for biologists who lack bioinformatics skills. For example, getting the raw read counts requires bioinformatics skills to map the RNA-seq data to a reference genome or transcriptome and generate read counts by using quantification tools such as Salmon, Kallisto and RSEM. The author should propose an easy-to-access method and provide step-by-step tutorials for biologists to generate the read counts from raw RNA-seq data, for example, using the Galaxy platform.  + It is true that data preparation can be quite complicated for non-specialists. Following the remark of the reviewer, we have added references and web links for two complete pipelines and tutorials for Galaxy on our DEVEA GitHub page (https://github.com/MiriamRiquelmeP/DEVEA/blob/main/Galaxy_tutorials.md). We have also included a brief explanation of them in the âUse caseâ section. - There are massive methods available for the function of differential expression (DE) analysis. Many comparison studies showed that the methods DESeq2, Limma-voom, edgeR and Sleuth have similar performance in DE analysis and each of these methods has a big user group. To attract more users to DEVEA, the author could highlight the advantages to use DESeq2 in DEVEA compared with other methods, such as Limma and edgeR, in the introduction or discussion. + It is correct that there are several methods available for RNA-seq differential expression analysis with comparable performances. We chose DESeq2 because it is widely used, very robust and recognised in the community. As suggested by the reviewer, we now stress, in the âOperationâ and briefly in the âConclusion and Future Perspectivesâ sections, the robustness of the method by mentioning an article that compares DESeq2's performance with other methods (Baik et al., PLoS One, 2020, 15(4):e0232271).     - In the tutorial, the table of contents is floating and overlaps with texts and videos. Is it possible to reallocate the position of the table of contents to avoid overlapping? + We have noticed that the HTML file that displays the tutorial is significantly heavy. As a result, some browsers do not render it properly and take some time to put each element in its place. In order to view the tutorial, as well as the complete workflow of DEVEA itself, we strongly recommend the use of Mozilla Firefox Web Browser. This part has been clarified in the âOperationâ section.      - In the example data link: https://github.com/MiriamRiquelmeP/DEVEA/tree/main/data, it would be better to include a readme file with descriptions of the example datasets. + A README.md file has been included on GitHub data folder, with all the test data inside (for Mus musculus and for Arabidopsis thaliana).      - âSupplemental-Information.txt: Running DEVEA locally on a machineâ: I would recommend the author build DEVEA as an R package. Then, instead of âDownload the compressed folder from the DEVEA GitHub repositoryâ, users can install and run DEVEA on a local machine as an R package. + In fact, DEVEA is a web interface with two R Shiny applications bundled together and due to R packages structural constraints, it is not so straightforward to package such an application. However, to facilitate the local installation of DEVEA, we have added a script on DEVEAâs github that will install all the necessary R dependences for DEVEA (script_libraryApp.R). Then the user only has to clone the DEVEA repository from github and launch the application (2-steps process) after running the script. For future versions, we plan to develop a Docker image, rather than an R package. We have detailed the process in the supplemental information (https://github.com/MiriamRiquelmeP/DEVEA/blob/main/Supplemental-Information.txt) - Page 3: âDespite the great progress to facilitate transcriptomics computational analyses, some improvements are still possible for these tools.â Although comparisons were given in the Related work section, the author should give brief descriptions of these tools and summarise their limitations as literature reviews in the introduction. The author also should address what are the possible improvements and why they are needed. + We have added a short paragraph in the introduction summarizing the features and limitations of the tools comparable to DEVEA in the introduction.   - Page 11: âReads were aligned on the mouse genome (ENSEMBL GRCm38 release 96) and a count matrix was generated.â For reproducibility, the tool for read mapping and count matrix generation should be referred to and cited. + The software tools we used for this task and further details are now indicated in the main text.   - Figure 7: the font size of labels and some details of the figures are too small. Would it be better to change the multiple plot layout to 3 rows x 2 columns? + We have tried to reformat the figure layout to 3 rows x 2 columns, but the result was really not satisfying, so we preferred to keep the original layout. The paper has quite a few typos. The author should carefully check the entire manuscript again. Here, I have picked out some of them:     Page 3: âkey steps of a complete RNA-seq analyses pipelineâ should be âkey steps of a complete RNA-seq analysis pipelineâ          Page 3: âdetailed bellow in the circular notesâ should be âdetailed below in the circular notesâ.          Page 5: âthe simple gene list (GL) can be build fromâ should be âthe simple gene list (GL) can be built fromâ          Page 6: âthe program extract the conditions and calculatesâ should be âthe program extracts the conditions and calculatesâ          Page 11: âTo demonstrate the usefulness of DEVEA, a RNA-seqâ should be âTo demonstrate the usefulness of DEVEA, an RNA-seqâ          Page 12: âwhich offers significantly less options sinceâ should be âwhich offers significantly fewer options sinceâ + We thank the reviewer for pointing out those typos. All of them have been corrected. We have also carefully proofread the manuscript for other remaining typos."
}
]
}
] | 1
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https://f1000research.com/articles/11-711
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https://f1000research.com/articles/12-332/v1
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24 Mar 23
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{
"type": "Research Article",
"title": "A comparative evaluation of micro shear bond strength and microleakage between the resin-modified glass ionomer cement and residual dentin following excavation of carious dentin using Carie CareTMÂ and conventional caries removal in primary teeth: an in vitro study",
"authors": [
"Megha Nair",
"Arathi Rao",
"Jayaprakash Kukkila",
"Srikant Natarajan",
"Suprabha Baranya Srikrishna",
"Megha Nair",
"Jayaprakash Kukkila",
"Srikant Natarajan",
"Suprabha Baranya Srikrishna"
],
"abstract": "Background: The bond between the dentin and restorative material contributes to the success of the restoration. Structural changes associated with prepared dentin may influence the bonding of restorative materials. The present study evaluates the bond between the resin-modified glass ionomer cement (RMGIC) and residual dentin following excavation of carious dentin using Carie CareTM and conventional caries removal in primary teeth. Methods: 52 primary teeth with dentinal caries were randomly grouped into group I, where caries removal was done using the conventional method, and group II which used Carie CareTM. All the teeth were restored using RMGIC. Micro shear bond strength between the residual dentin and the cement was tested using universal testing machine and the dye penetration method was used for microleakage testing. Independent t-test was performed for intergroup comparison. Pearson chi-square test was carried out to evaluate the microleakage patterns in the enamel and dentin. Results: The mean micro-shear bond strength of group I was 6.03±1.6 and that of group II was 8.54±2.92; this difference was statistically significant with a p-value of 0.012. Microleakage was higher in the test group (1.38±0.51) than the control group (0.77±0.6) and was significant with a p-value of .036. Conclusions:\nPapain-based chemomechanical agent Carie CareTM can be used as an alternative method to conventional caries removal. However, further studies need to explore methods to improve the marginal sealing capacity of RMGIC to the residual dentin after chemomechanical caries removal.",
"keywords": [
"Carie CareTM",
"Primary teeth",
"residual dentin",
"resin modified glass iononmer"
],
"content": "Introduction\n\nConventional caries removal method using rotary burs is easy and quick but has also been associated with unnecessary removal of affected dentin that could have been remineralized, patient discomfort and pain that may necessitate an administration of local anaesthesia.1\n\nTo overcome these shortcomings, the chemomechanical caries removal (CMCR) system was introduced, forming the foundation of minimally invasive caries removal techniques. Carie CareTM is one such formulation containing purified papain enzyme. It was introduced in India by Vittal Mallya Scientific Research Foundation, Bengaluru, Karnataka, India, and Uni-Biotech Pharmaceuticals Pvt. Ltd in 2011.2,3\n\nThe bond between the dentin and restorative material contributes to the success of the restoration. Structural changes associated with prepared dentin may influence the bonding of restorative materials and most of the studies have established that CMCR produces a roughened surface with altered hardness.4,5\n\nGlass ionomer cement is still the preferred restorative material for restoring primary teeth, but there are no studies evaluating the bond between the glass ionomer cement with the residual dentin following caries removal using Carie CareTM. The present study was thus planned to evaluate the bond between the resin-modified glass ionomer cement and residual dentin following excavation of carious dentin using Carie CareTM and conventional caries removal in primary teeth.\n\n\nMethods\n\nThe study was conducted in the Department of Paediatric and Preventive Dentistry and the Department of Oral Pathology, Manipal College of Dental Sciences, and the Department of Dental Materials at Yenepoya Dental College.\n\nThe present study was an experimental in vitro study, designed according to the modified Consolidated Standards of Reporting Trials (CONSORT).\n\nAll procedures were performed in conformity with the ethical standards of the institutional Ethics committee, Manipal College of Dental Sciences, Mangalore. Ethical clearance was obtained from the Institutional Ethics Committee (ref: 19085 dated 10th October 2019) before the study. All the collected teeth were grouped and could not be traced back to any person/child.\n\nThe sample size was calculated to be 52 (n=26 in each group) at 90% power, 5% alpha error, and a clinically significant difference of 1 unit.\n\n52 freshly extracted human primary first and second molars with class I or class II cavitated dentinal lesions with sufficient opening for hand instrumentation were selected for this study. The teeth were selected based on the inclusion and exclusion criteria and were indicated for extraction from patients attending the clinics for dental treatment at the department of Pediatric and Preventive Dentistry, Manipal College of Dental Sciences, Mangalore.\n\nExclusion criteria included teeth with caries involving the pulp, crack, or defect on the enamel surface.\n\nAll the selected teeth were cleaned thoroughly with hand scalers and fluoride-free pumice to remove the extrinsic deposits and blood. The teeth were then stored in 0.1% thymol solution.3,4\n\nPrimary outcome:\n\n1. The bonded interface between resin-modified glass ionomer cement and the treated tooth surface was subjected to modified short-beam shear (MSBS) testing. The micro shear bond strength was calculated using the formula: Micro shear bond strength (MPa)=Shear Force(N)/Cross-sectional area (mm2).\n\n2. Microleakage analyses were made by observing the penetration of the dye into the tooth surface through the interface.\n\nSecondary outcome\n\n1. The extent of dye penetration into the enamel and/or dentin was noted and recorded.\n\nBased on the inclusion and exclusion criteria, a total of 52 teeth were selected out of the collected 70 teeth (Figure 1). They were randomly divided into two groups (n=26 teeth in each group) by chit method as follows:\n\n1. Group I â Control group: Conventional caries removal.\n\n2. Group II â Test group: Caries removal using Carie CareTM\n\nEach group was further randomly divided into two subsets (n=13 in each group) using the same randomization technique as follows:\n\n1. Group I and II A â For micro shear bond strength testing.\n\n2. Group I and II B â For microleakage testing.\n\nThe random allocation sequence and enrolment of samples were done by a person not involved in the study. The person who tested and evaluated both micro shear bond strength and microleakage and the statistician who carried out the analysis were blinded to the allocation of the samples. However, the operator who had carried out the restorative procedures was not blinded to the allocation.\n\nCaries removal\n\nA. Caries removal by the conventional method\n\nCaries was removed by a single operator using a slow-speed contra angled handpiece with large round diamond bur (NMD Nexus Medodent Dental Contra Angle Low Speed Handpiece (Latch Type) and 001/018 round bur) under cooling until all the infected dentin was removed. The completeness of the caries removal was checked by running a sharp explorer tip on the floor of the cavity. It should neither give any tug-back sensation nor should stick to the dentin. The caries removal was continued until the criteria were satisfied.6\n\nB. Caries removal using Carie CareTM\n\nThe gel was placed on the cavitated lesions via the syringe in which it is dispensed. It was left untouched to allow it to work for 60 seconds. When the gel turned cloudy, the softened dentin and the gel were removed using a spoon-shaped hand excavator without applying pressure. The process was repeated until the gel no longer turned cloudy. The completeness of the caries removal was assessed by using the same criteria as for the first group.3\n\nRestoration of the teeth\n\nA dentin conditioner (10% polyacrylic acid) was placed on the exposed dentin surface using a micro brush. A PVC tube (internal diameter of about 0.9 mm and 2 mm height) was placed on the dentin conditioner and cured for 10 seconds. RMGIC (GC Gold Label Light Cured Universal Restorative Material, GC Corporation, Tokyo, Japan) was packed compactly inside the tube using a plastic filling instrument, avoiding any voids. It was then cured for 20 seconds using a visible light curing device (Elipar 2500, 3M ESPE, Dental Products, St Paul, MN, USA). A radiometer (Demetron 100, Demetron Research Corp, USA) was used to verify the light intensity of the halogen light-curing device (minimum threshold = 600 mW/cm2). The completely set specimens were stored in distilled water for 24 hours.3\n\nSample preparation for micro shear bond strength testing\n\nTeeth were sectioned at the level of cementoenamel junction to remove the remaining roots with the help of a high-speed handpiece and diamond burs. The specimens were then placed on a glass slide. Two L-shaped molds that when put together created a rectangular space and were used for the fabrication of resin blocks around the specimens. Once the resin was set, the glass slide along with the sticky wax was removed. The resin blocks were then trimmed and polished with 400 and 600-grit silicon carbide (SiC) papers to the desired dimension. The dimensions of the resin block to fit the testing tool jig were approximately 28 mm high, 13 mm wide, and 10 mm thick (Figure 2A, B)3\n\nSample preparation for microleakage\n\nThe teeth were coated with a single layer of air-dry nail varnish (Lakme, India) except at an area approximately 2 mm around the periphery of the restoration. The cervical portion of the teeth was sealed with sticky wax to prevent the seeping of the dye through the cervical aspect. The teeth were placed in 2% methylene blue (Merck KGa A-C.I.52015) for 24 h at room temperature. They were then removed and washed under running water. The teeth were then sectioned in the buccolingual direction using a diamond disc to visualize the penetration of dye at the restoration tooth interface.7\n\nMicroshear bond strength testing\n\nTesting for micro shear bond strength was done using a universal testing machine (Type: HPBSD, Model no: TSI-BSD-20KN, Serial no: 170710). The samples were fixed onto the jig which in turn was fixed on the mechanical jaw of the micro shear universal testing tool. The bonded interface was then tested using a chisel at a crosshead speed of 1.0 mm/min.7\n\nMicroleakage testing\n\nThe degree of dye penetration was scored using a stereomicroscope (ZTX-3E, China) at X20 magnification. The score which was higher was taken as the score for that particular tooth.\n\nThe following scoring criteria were used8 (Figure 3):\n\n0 â No dye penetration\n\n1 â Dye penetration into enamel only\n\n2 â Dye penetration into the enamel and dentin\n\n3 â Dye penetration into pulp.\n\nAfter testing both groups for micro shear bond strength and microleakage, the data was entered and analyzed using Statistical Package for Social Science (SPSS), version 20 (SPSS Inc.). An Independent t-test was performed for comparing the mean values of micro shear bond strength and microleakage between the conventional and Carie CareTM groups. Dye extended into the tooth structure at the restoration interface. The extent of the dye into the enamel/dentin was evaluated using the Pearson chi-square test.\n\n\nResults\n\nA total of 52 teeth were divided equally into group I and group II to evaluate micro shear bond strength and microleakage between the resin-modified glass ionomer cement and residual dentin following excavation of carious dentin using Carie CareTM and by conventional caries removal in primary teeth.\n\nPrimary outcome\n\nThe highest scores were 10.16 MPa and 14.84 MPa in conventional caries removal and Carie CareTM groups respectively, and the lowest score in the conventional caries removal group was 4.08 MPa while it was 3.53 MPa in the Carie CareTM group. The average micro shear bond strength score was 6.03±1.6 MPa in the conventional caries removal group and 8.54±2.92 MPa for the Carie CareTM group.\n\nIndependent t-test revealed a t value of -2.706 and the difference in the mean micro-shear bond strength values between both the groups was statistically significant with a p-value of 0.012 (Table 1).\n\nPrimary outcome\n\nMicroleakage was seen in all the samples of the Carie CareTM group, while 69.20% of the samples in the conventional caries removal group exhibited microleakage. The mean microleakage value of the conventional caries removal group was 0.77±0.6 and that of the Carie CareTM group was 1.38±0.51.\n\nIndependent t-test revealed a t value of -2.828 and the difference in the microleakage values between both the groups was statistically significant with a p-value of 0.009 (Table 1).\n\nSecondary outcome\n\nOut of the 9 samples with microleakage in the control group, 8 had leakage into enamel and 1 into enamel and dentin. In group II, 8 samples exhibited microleakage into the enamel, and 5 into enamel and dentin. The microleakage patterns into the enamel and dentin were found to be statistically significant with a p-value of .036 (Table 2).\n\n\nDiscussion\n\nThe chemo-mechanical caries removal (CMCR) method makes use of a chemical that softens the degraded collagen fibers in the infected dentin which is then easily removed by gentle mechanical action by a hand instrument without affecting the healthy tissues. There are two types of chemomechanical caries removal agents, sodium hypochlorite-based and papain enzyme based. Papain is a proteolytic enzyme, derived from the latex of the papaya leaves and fruit with bactericidal, bacteriostatic as well as anti-inflammatory properties similar to the actions of the human pepsin enzyme. It acts as a debriding agent and doesn't impair healthy tissues. Examples of this system are Papacárie ® and Carie CareTM. Carie CareTM is used in the present study and is relatively new, simple to use and does not require any training or any special equipment for its use, and is much more economical. Other components of Carie CareTM are chloramine, gelling agents and clove oil, colored gel (blue), sodium chloride, and sodium methylparaben.3 Chloramines help in the healing process and shorten tissue repair time and have the potential to dissolve carious dentin through chlorination of partially degraded collagen. This helps in the disruption of collagen structure, dissolves hydrogen bonds, and helps in tissue removal. Clove oil has an analgesic and antiseptic action. Sodium methylparaben is used as a preservative.9â12\n\nAnwar et al.5 in their study found that the microhardness (KHN) of the residual dentin following Carie CareTM application was reduced compared to that following caries removal using burs.\n\nThere is no existing literature evaluating the bond between resin-modified glass ionomer cement and residual dentin treated with a papain-based CMCR agent, Carie CareTM in primary teeth. The most cited failures of restoration are lack of marginal adaptation and loss of retention.13 RMGIC was chosen in the present study as it is the most preferred material for the restoration of primary teeth.14\n\nThe present study was thus initiated to evaluate the bond between the residual dentin and resin modified glass ionomer cement following caries removal with Carie CareTM and the conventional method.\n\nIn vitro tests have many advantages such as simplicity, ease of sampling for microleakage, etc. Shear bond strength testing is considered to be one of the most commonly used methods for testing bond strength, especially for any substrate susceptible to crack propagation during sample preparation like glass ionomer cements.15 Micro shear bond strength testing was used in the present study as it results in a uniform stress distribution over a small area (<1 mm2) leading to more reliable results.16\n\nThe current study demonstrated that the micro shear bond strength between Carie CareTM treated residual dentin and RMGIC was significantly higher than the conventional caries removal group. This finding was different from that of other studies which were done on permanent teeth.3 Many earlier studies on permanent teeth using an earlier system of CMCR showed that the chemomechanical method did not influence the bond strength.3,17\n\nCaries removal using low-speed rotary instruments produced a smooth and uniform smear layer over the dentin surface while the dentin exhibited intertubular microporosity with minimal or no smear layer, exposing the dentinal tubules following the use of Carie CareTM with fibrous structure inside the tubules in primary molars. The presence of open dentinal tubules in chemo-mechanical caries removal is attributed to the initial high pH of the gel due to the presence of chloramine.18\n\nBonding could also vary depending on the orientation and density of the dentinal tubules. The infiltration of the restorative material is higher in the deeper regions of dentin because of the wider dentinal tubules and perpendicular orientation of the tubules to the pulp wall. Likewise, the bonding in dentin is better in the proximal walls as compared to the occlusal wall.19 These factors could explain the differing results of other studies compared to this study.\n\nThe rationale of testing microleakage is that it can be considered as a proxy for the penetration of bacteria and fluids along the restoration-tooth interface intraorally which may result in hypersensitivity, secondary caries, pulpitis, etc. There are several methods of evaluating microleakage and one of them is the dye penetration method using dyes like methylene blue, rhodamine, or erythrosine. It is simple, inexpensive, doesn't require the use of complex chemicals and testing equipment, and also allows the investigator to view the longitudinal sections but in a two-dimensional view.20\n\nIn the current study, the mean microleakage values in the Carie CareTM group were higher compared to the conventional caries removal group and extended into the dentin, which is similar to other studies.21,22\n\nKhattab & Omar et al.21 concluded that glass ionomer exhibited more microleakage and lower micro shear bond strength than composite resin restoration after the use of Papacarié gel in primary teeth.\n\nCarie CareTM was found to be easy to handle, easy and efficient for caries removal, and provides good bond strength with resin-modified glass ionomer cement. The drawback of Carie CareTM is that it provides no improvement in microleakage which is one of the main drawbacks of CMCR agents.\n\nThe following conclusions can be drawn from the present study:\n\n1. The mean micro shear bond strength following carious removal using Carie CareTM was found to be better compared to the control group in which caries were removed by the conventional method.\n\n2. Carie CareTM exhibited more microleakage compared to that of the control group and extended into the enamel and dentin.\n\n\n\n1. In the present study all primary teeth with class I or class II caries were selected. The depth of the carious lesion, the lesion activity, the shape and location of the lesions, and the consistency of the dentin could not be standardized, which could have influenced the results.\n\n2. Extracted teeth may respond very erratically to the caries excavation compared to vital teeth, because of the outward flow of dentinal fluid in the tubules in vital teeth. The future scope of the present study may be to observe structural changes in the dentin of primary teeth following caries removal using Carie CareTM to obtain more insight.\n\nKey points\n\n\n\n⢠Carie CareTM may be a better choice over other chemomechanical caries removal systems as they do not affect the bond strength of restorative material.\n\n⢠Microleakage is associated with Carie CareTM similar to other chemomechanical caries removal systems.",
"appendix": "Data availability\n\nfigshare: Microshear bond strength Raw Data, https://doi.org/10.6084/m9.figshare.22213999.v1. 23\n\nfigshare: Raw Data-Microleakage, https://doi.org/10.6084/m9.figshare.22214011.v1. 24\n\nfigshare: Figure 1. https://doi.org/10.6084/m9.figshare.22140266.v1. 25\n\nfigshare: Figure 2. https://doi.org/10.6084/m9.figshare.22140293.v2. 26\n\nfigshare: Figure 3. https://doi.org/10.6084/m9.figshare.22140296.v3. 27\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nfigshare: Modified CONSORT checklist for reporting in vitro studies of dental materials for âA comparative evaluation of micro shear bond strength and microleakage between the resin-modified glass ionomer cement and residual dentin following excavation of carious dentin using Carie CareTM and conventional caries removal in primary teeth: an in vitro studyâ. https://doi.org/10.6084/m9.figshare.22140305.v1.\n\n\nReferences\n\nNair S, Nadig RR, Pai VS, et al.: Effect of a Papain-based Chemomechanical Agent on Structure of Dentin and Bond Strength: An in vitro Study. Int. J. Clin. Pediatr. Dent. 2018; 11(3): 161â166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarie-Care - a chemo-mechanical agent for removal of caries: Accessed on September 2021. Reference Source\n\nChittem J, Sajjan GS, Varma KM: Comparative evaluation of micro-shear bond strength of the caries-affected dentinal surface treated with conventional method and chemo-mechanical method (papain). J. Conserv. Dent. 2015; 18(5): 369â373. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElkateb M, El Meligy O, Kotb R, et al.: Dentin Topographic Features following Chemomechanical Caries Removal in Primary Teeth. J. Clin. Pediatr. Dent. 2016; 40(6): 472â479. PubMed Abstract | Publisher Full Text\n\nAnwar AS, Kumar RK, Prasad Rao VA, et al.: Evaluation of microhardness of residual dentin in primary molars following caries removal with conventional and chemomechanical techniques: An in vitro Study. J. Pharm. Bioallied Sci. 2017; 9(5): S166âS172. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEricson D, Zimmerman M, Raber H, et al.: Clinical evaluation of efficacy and safety of a new method for chemo-mechanical removal of caries: A multi-centre study. Caries Res. 1999; 33: 171â177. PubMed Abstract | Publisher Full Text\n\nIsmail AM, Bourauel C, ElBanna A, et al.: Micro versus Macro Shear Bond Strength Testing of Dentin-Composite Interface Using Chisel and Wireloop Loading Techniques. Dent. J (Basel). 2021; 9(12): 140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrabhakar AR, Madan M, Raju OS: The marginal seal of a flowable composite, an injectable resin modified glass ionomer and a compomer in primary molarsâan in vitro study. J. Indian Soc. Pedod. Prev. Dent. 2003 Jun; 21(2): 45â48. PubMed Abstract\n\nTonami K, Araki K, Mataki S, et al.: Effects of chloramines and sodium hypochlorite on carious dentin. J. Med. Dent. Sci. 2003; 50(2): 139â146. PubMed Abstract\n\nBarwart O, Moschen I, Graber A, et al.: Invitro study to compare the efficacy of N-monochloro-D, L-2-aminobutyrate (NMAB, GK-101E) and water in caries removal. J. Oral Rehabil. 1991; 18(3): 523â529. PubMed Abstract | Publisher Full Text\n\nYazici AR, Atilla P, Ãzgunaltay G, et al.: In vitro comparison of the efficacy of Carisolv and conventional rotary instrument in caries removal. J. Oral Rehabil. 2003; 30(3): 1177â1182. PubMed Abstract | Publisher Full Text\n\nRamamoorthi S, Nivedhitha M, Vanajassun PP: Effect of two different chemomechanical caries removal agents on dentin microhardness: An in vitro study. J. Conserv. Dent. 2013; 16(5): 429â433. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDe Munck J, Van Landuyt K, Peumans M, et al.: A critical review of the durability of adhesion to tooth tissue: Methods and results. J. Dent. Res. 2005; 84(2): 118â132. PubMed Abstract | Publisher Full Text\n\nQvist V, Manscher E, Teglers PT: Resin-modified and conventional glass ionomer restorations in primary teeth: 8-year results. J. Dent. 2004; 32(4): 285â294. PubMed Abstract | Publisher Full Text\n\nArmstrong S, Geraldeli S, Maia R, et al.: Adhesion to tooth structure: a critical review of âmicroâ bond strength test methods. Dent. Mater. 2010; 26(2): e50âe62. PubMed Abstract | Publisher Full Text\n\nPlacido E, Meira JB, Lima RG, et al.: Shear versus micro-shear bond strength test: a finite element stress analysis. Dent. Mater. 2007 Sep; 23(9): 1086â1092. PubMed Abstract | Publisher Full Text\n\nBurke FM, Lynch E: Glass polyalkenoate bond strength to dentine after chemo mechanical caries removal. J. Dent. 1994; 22(5): 283â291. PubMed Abstract | Publisher Full Text\n\nCorrêa FN, Rodrigues Filho LE, Rodrigues CR: Evaluation of residual dentin after conventional and chemomechanical caries removal using SEM. J. Clin. Pediatr. Dent. 2018; 32(2): 115â120. PubMed Abstract | Publisher Full Text\n\nMontagner AF, Carvalho MP, Susin AH: Microshear bonding effectiveness of different dentin regions. Indian J. Dent. Res. 2015; 26(2): 131â135. PubMed Abstract | Publisher Full Text\n\nAlHabdan AA: Review of microleakage evaluation tools. J. Int. Oral Health. 2017; 9(4): 141â145. Publisher Full Text\n\nKhattab NM, Omar OA: PapainâBased Gel for Chemo-Mechanical Caries Removal: Influence on Microleakage and Microshear Bond Strength of Esthetic Restorative Materials. J. Am. Sci. 2012; 5(3): 67â69. Publisher Full Text\n\nKitsahawong K, Seminario AL, Pungchanchaikul P, et al.: Chemomechanical versus drilling methods for caries removal: an in vitro study. Braz. Oral Res. 2015; 29: 1â8. Publisher Full Text\n\nNair M, Rao A, Kukkila J, et al.: Micro shear bond strength -raw data. [Dataset]. figshare. 2023. Publisher Full Text\n\nNair M, Rao A, Kukkila J, et al.: Microleakage - Raw Data. [Dataset]. figshare. 2023. Publisher Full Text\n\nRao A, Nair M, Kukkila J, et al.: Figure 1. figshare. Figure.2023. Publisher Full Text\n\nNair M, Rao A, Kukkila J, et al.: Figure 2. figshare. Figure.2023. Publisher Full Text\n\nRao A, Nair M, Kukkila J, et al.: Figure 3. figshare. Figure.2023. Publisher Full Text"
}
|
[
{
"id": "168089",
"date": "10 May 2023",
"name": "Parajeeta Dikshit",
"expertise": [
"Reviewer Expertise Pediatric Dentistry",
"Child Psychology",
"Dental Caries",
"Dental Materials",
"Gingival diseases",
"Behaviour Management",
"Orthodontics in Children",
"Pediatric Endodontics",
"Trauma Management of Anterior teeth."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is written excellently, and is well organized with detailed descriptions in all sections. The article addresses an important topic that has been minimally researched and will be an important milestone for research in dentistry. Based on my level of expertise the article meets the criteria to be published with minimal revisions.\nFollowing are my comments and suggestions after reviewing the allotted article.\nAbstract\nThe abstract has summarized the research adequately and does not have scope for any more revision. The methods section in abstract begins with a number which can be rectified by mentioning the number in words. The abstract could have a clearer conclusion.\nIntroduction\nThe introduction is brief encompassing the highlights of what to expect from the present study. It has mentioned about the materials and techniques used adequately. The authors are suggested to mention about the history of chemomechanical caries removal technique with special mention about the type of glass ionomer cements used in primary teeth and why Resin modified Glass ionomer cements are preferred over the others.\nMaterials and Methods\nThis section has been meticulously written describing each step in detail which will be an asset for future research. There are with only few minor suggestions as mentioned below:\nThe authors are suggested to mention the age group of children whose teeth were included in the study\n\nThe time period for storage of teeth after extraction and before preparation is suggested to be mentioned.\n\nThere could have also been a mention about the criteria for teeth selection based on range of depth of the caries lesion with classification of it.\n\nResults\nThe result can be published as it is as the data has been analyzed well and described with necessary tables.\n\nDiscussion\nThe article has sufficiently discussed all aspects of the research with the comparison of materials used as well as the technique. Cross references with similar as well as contrasting research has been appropriately described by the authors. This study is an initiative to evaluate the bond between residual dentin and resin modified glass ionomer cement which will be a landmark for future research in this category. The limitations have been mentioned by authors for future studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "169584",
"date": "23 May 2023",
"name": "Kushal Vasanth Shetty",
"expertise": [
"Reviewer Expertise Dental research"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic of the study has been put forth well in the article. Newer methods of caries excavation have been practiced to avoid excess unnecessary removal of the sound dentin. The study has been done meticulously and written excellently. At the same time, the author has addressed the needed concern- will the residual dentin bond adequately with the restorative material and for the same reasons based on my level of expertise, I suggest that the article meets the criteria to be indexed.\nFollowing are my comments after reviewing the article.\nAbstract The abstract is well structured and is summarized adequately. It is precise and does not need any revision\nIntroduction The authors have clearly mentioned the purpose of the study with clear background. It is brief and gives a clear introduction for further reading. Most of the studies are performed on permanent teeth, Since glass ionomer cement is the most frequently used restorative material in primary teeth, choice of the material is the need of the time especially when used following a newer chemomechanical caries removal system in a primary teeth.\n\nMaterials and Methods The method described is self explanatory and very easy to reproduce which should be the hallmark of any study. The sectioned mentioned in the materials and method makes it clear for any reader to understand. Some of the mentions in the article such as the anonymity aspect of the extracted teeth, primary and secondary outcomes, randomization and blinding are the positive observations,\nAuthors could have mentioned the period for which the teeth were stored before caries removal.\nResults The results are clearly mentioned and explained with tables.\n\nDiscussion The authors have briefly mentioned the types of CMCR with clear explanation of the current study material and its components. Reasons for choosing the restoration material in a primary material is well explained. Authors have also included the discussion on the rationale for choosing the different tests used.\nThe outcome of the study is discussed with studies having same and contradictory outcomes.\nBy mentioning the limitations of the study, authors have hinted at the scope for the improvement of the study. Key points suggests to the researcher that future study is needed in CMCR to improve its properties and minimizing microleakage.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-332
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https://f1000research.com/articles/11-1551/v1
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21 Dec 22
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{
"type": "Systematic Review",
"title": "Barriers to compliance with evidence-based guidelines for ventilator-associated pneumonia among critical care nurses: A scoping review",
"authors": [
"Muna Al-Tamimi",
"Fatma Refaat",
"Wegdan Bani Issa",
"Fatma Refaat",
"Wegdan Bani Issa"
],
"abstract": "Background: Healthcare organizations provide evidence-based guidelines designed to support nurses in preventing ventilator-associated pneumonia (VAP) in intensive care units (ICUs), but there are barriers to compliance with such guidelines. This review explicitly explored evidence of compliance barriers among critical care nurses. Methods: A systematic search was conducted in PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), and EBSCO databases for relevant English-language studies published between January 2003 and June 2022, focused on barriers to nursing compliance with VAP prevention guidelines. Data was reported according to the Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMAScR) guidelines. Results:\n230 publications were screened, resulting in 53 full-text articles being retrieved after removing duplicates, of which 13 relevant to the aims of the review and meeting the inclusion criteria were included for data extraction. One was a qualitative study, while the remainder were quantitative. Simple descriptive content analysis identified the barriers to critical care nursesâ compliance with VAP prevention guidelines, and categorized them as: (1) work environment barriers (e.g., lack of equipment and supplies; lack of staff and time; lack of educational support; and ineffective supportive system); (2) nurse-related barriers (limited personal competencies); and (3) situation-related barriers (patient health, discomfort, and adverse events). Conclusions: This review revealed important evidence on barriers to VAP prevention guidelines compliance. Nurses are challenged mainly by work-environmental barriers along, with the presence of nurse and situational barriers. It is evident from the findings that further qualitative and mixed-methodology follow-up studies are recommended to further explore the issues in depth. Healthcare leaders must be aware of these barriers and integrate work policies that assist in overcoming them, to increase compliance.",
"keywords": [
"Barriers",
"Compliance",
"Critical care nurses",
"Ventilator-associated pneumonia",
"Prevention guidelines"
],
"content": "Introduction\n\nVentilator-associated pneumonia (VAP) is a common and arduous respiratory infection that targets mechanically ventilated patients (MVPs) in intensive care units (ICUs) (Hunter, 2012; Metersky & Kalil, 2018; Valles et al., 1995). VAP is characterized as an infection of the pulmonary parenchyma that appears after at least 48 hours of endotracheal intubation for mechanical ventilation, with day 1 being the day of ventilator insertion. It causes about a third of nosocomial pneumonia cases in ICUs (Torres et al., 2017). According to a Centers for Disease Control and Prevention (CDC) report, pneumonia was the most common infection in US acute care hospitals in 2015, with 32% occurring as a result of the use of ventilators (Magill et al., 2018). Another study conducted across three Gulf Cooperation Council countries (Bahrain, Oman, and Saudi Arabia) estimated that the local VAP rate was 217% higher than in the US, according to National Healthcare Safety Network data (El-Saed et al., 2016). Ventilated patients who develop VAP suffer higher mortality than ventilated patients who do not (Ibrahim et al., 2001; Safdar et al., 2005). Moreover, VAP substantially increases ventilator time and length of ICU stay (Papazian et al., 2020), along with ICU care costs (Warren et al., 2003; Zimlichman et al., 2013).\n\nAs a result of the high mortality, morbidity, and hospital expenses associated with VAP, a number of guidelines have been published since the 1980s to prevent and control it (Rello et al., 2002; Society & America, 2005). Efforts continued by health organizations and societies such as the European Centre for Disease Prevention and Control (ECDC) and the CDC to set and update evidence-based guidelines (EBGs) and strategies that markedly proved to minimize the occurrence of VAP and improve outcomes, and the quality of care (QoC) delivered to MVPs ((IHI). 2012; Klompas et al., 2022; Torres et al., 2017).\n\nNurses play a pivotal role in providing safe and direct care to MVPs, including practicing VAP prevention strategies that essential to prevent infection and therefore improve the quality of patient care and outcomes in ICUs (Jansson et al., 2013; Osti et al., 2017)\n\nAccording to Pogorzelska et al. (2011), nursing compliance with VAP prevention guidelines in ICUs (hereinafter âNCâ) should be at least 95%, in order to effectively reduce VAP incidence. Therefore, strict compliance with strategies and recommendations is required from nurses, who must appropriately perform several interventions and procedures to achieve optimum outcomes (Miranda da Cruz & da Silva Martins, 2019; Rello et al., 2013; Tabaeian et al., 2017).\n\nFacilitating and promoting changes in patient care, and encouraging NC is crucial for VAP prevention success, and also providing regular feedback on process measure performance and outcome rates are among the best practices to facilitate adherence with guidelines (Crunden et al., 2005; Klompas, 2017).\n\nIn spite of the existence of clinical practice guidelines for preventing VAP, these guidelines are not consistently followed (Cason et al., 2007; Ricart et al., 2003). Reported levels of compliance with and proper use of strategies vary widely in various contexts, across health systems, specialties, and nurses, ranging from 20% to nearly 100% (Beattie et al., 2012; Bird et al., 2010; Rello et al., 2002). Several studies showed that nurses generally exhibit low NC, which could be attributable to diverse factors (Aloush et al., 2018; Aloush & Al-Rawajfa, 2020; Jahansefat et al., 2016; Jam et al., 2018). Given the critical impact of NC on patientsâ QoC, it is essential to identify the factors influencing it that might impede the proper implementation of EBGs, and hence affect patient outcomes.\n\nThere are few existing reviews of studies reporting NC barriers (hereinafter âNCBsâ). Consequently, it is vital to comprehensively map the evidence on relating to the findings available in this topic, how studies have been conducted, the key characteristics of studies, and important knowledge gaps. A scoping review is used to explore the breadth or extent of the literature, map and summarize the evidence, and inform future research (Tricco et al., 2016). This scoping review aimed to show the available evidence of the barriers toward critical care nursesâ (CCNs) NC. The primary objective of this scoping review is to understand the types and extent of evidence available in relation to NCBs. The detailed aims of this review were to: (1) examine the characteristics of studies that have reported the barriers toward preventing VAP guidelines among CCNs; (2) identify and summarize key findings of related studies; and (3) identify gaps of extant studies that may help inform future research in this area.\n\n\nMethods\n\nThe scoping review was conducted in accordance with the Joanna Briggs Institute (JBI) methodology to determine CCNsâ NCBs for VAP EBGs. It followed the frameworks of Arksey and O'Malley (2005) and Levac et al. (2010) that have underpinned the development of the JBI approach for conducting scoping reviews (Peters et al., 2015). Moreover, It is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) to provide a guidelines to the review (Tricco et al., 2018). Population-Concept-Context (PCC) elements is followed in line with JBIâs recommendations for scoping reviews, in order to guide the inclusion criteria development, facilitate literature searches, and offer a robust framework of this scoping review (Peters et al., 2020).\n\nThis scoping review examined the following research question: What are the barriers to compliance with the VAP prevention guidelines among CCNs. The population comprised nurses practicing in any country. The core concept examined was the barriers to compliance with VAP guidelines prevention and the context was ICUs.\n\nThe search was performed between March-June 2022 to include relevant studies in English language only (excluding non-English language studies as they required translation), using the electronic research databases Cumulative Index to Nursing and Allied Health Literature (CINAHL), PubMed, and EBSCO. Database searches were targeted to full-text, peer-reviewed articles, including primary research and any type of review. Relevant articles selected for review were published between January 2003 and June 2022, to include studies since the issuance of the CDCâs updated recommendation âGuidelines for Preventing Health-Care--Associated Pneumoniaâ (Tablan et al., 2003). Data were collected using the main keywords on VAP, Prevention guidelines, Barriers, Compliance, Intensive care, and Nurses. The detailed search strategy is available as additional file in the data availability. The participants of the included studies had to be CCNs and charge nurses who had worked directly with MVPs, with responsibility for implementing the VAP prevention guidelines (hereinafter âVAP PGsâ) for which they were reporting the perceived NCBs. Assistant nurses, nurse managers and other health professionals were excluded. The focus of studies had to be identifying barriers to NC with VAP PGs. Other factors that were irrelevant or which did not hinder NC were excluded. The included studies had to have been conducted in ICUs using mechanical ventilation for patients. Table 1 summarizes the inclusion and exclusion criteria for the selected studies.\n\nThe search results were collated, uploaded to EndNote X9.3.3/2008 (RRID:SCR_014001; Clarivate Analytics, PA, USA), and duplicates were removed. Three reviewers (MA, FR, and WB) independently screened titles and abstracts to determine whether they met inclusion criteria. Relevant sources were retrieved in full. A hand search of the bibliographies of initially included studies was undertaken in order to identify additional relevant works not gleaned from the database search (Peters et al., 2020). All reviewers independently examined the full texts of selected citations to ensure that they met the inclusion criteria, and evidence sources that did not meet the inclusion criteria were excluded. During the selection process, disagreements between the reviewers were resolved through discussion, as detailed in the Results section.\n\nData were extracted from the articles by the reviewers using JBI data extraction tool. Detailed information about the authors, years of publications, country, study aims, study methods, and key findings relevant to the review aim/s were extracted (Table 2). The data extraction tool was revised and modified by all authors as necessary during the process of extracting data from each evidence source included in this review. A discussion was used to resolve disagreements between reviewers. There was no need to contact the authors of the articles due to the comprehensiveness of reported data (i.e., no missing data).\n\nThe relevant literature was summarized in separate tables based on the review aims. For this review, a critical appraisal of individual sources of evidence was not necessary (Peters et al., 2020). All authors independently reviewed the studies of NCBs in ICUs and extracted the barriers described in the results sections of the included studies. They were subsequently grouped into categories and subcategories, based on the identified contexts of barriers, using basic descriptive content analysis, which is an optimal method to summarize findings (Elo & KyngÀs, 2008). The principles of inductive content analysis were used to analyze, categorize, and quantify NCBs for VAP prevention. After an initial open-coded base, similar open-codes were grouped together into thematic categories, each of which was labelled using content-specific keywords and subcategories.\n\n\nResults\n\nThe initial search of this review identified 400 articles. A total of 230 non-duplicate records were subsequently identified through the searching of databases and the reference lists of included articles. After screening titles and abstracts (objectives of the articles) for relevancy against inclusion criteria, 53 articles were identified and retrieved for full-text data extraction and screening. Of these, 53 articles, 13 met all inclusion criteria and were included in this review. The flow chart (Figure 1) of the review decision process was adapted from the PRISMA flowchart (Page et al., 2021).\n\nOf the 13 included studies (as shown in Table 3), one was qualitative (Atashi et al., 2018) and the remainder quantitative. The included studies were published between 2003 and 2022, with the following numbers of study per country: two (15.4%) in Finland (Jansson et al., 2013, 2018); three (23%) in Iran (Atashi et al., 2018; Dehghan et al., 2022; Yeganeh et al., 2019); two (15.4%) in Jordan (Aloush & Al-Rawajfa, 2020; Hassan & Wahsheh, 2017); one (7.7%) in Jordan, Egypt, and Saudi Arabia (âMiddle Eastâ) (Aloush et al., 2018); one (7.7%) in Spain (Ricart et al., 2003); two (15.4%) in Saudi Arabia (Al-Sayaghi, 2021; Yaseen & Salameh, 2015); one (7.7%) in Tanzania (Bankanie et al., 2021); and one (7.7%) in the USA (Kiyoshi-Teo et al., 2014).\n\nThis review yielded evidence for variety of NCBs, categorized into barriers relating to the âwork-environmentâ, ânurseâ, and âsituationâ. These barriers were further categorized into subcategories of barriers: work environment (lack of equipment and supplies; lack of staff and time; lack of educational support; and ineffective supportive system), nurse (limited personal competencies), and situation (patient health, discomfort, and adverse events) (Table 4).\n\n\nDiscussion\n\nThis review aimed to examine the characteristics and conclusions of studies to identify barriers confronting nurses, thwarting their compliance with EBGs for VAP prevention in ICUs. It is apparent that there has been an increased rate of publication of studies assessing NCBs over the past five years (during which nine out of the 13 identified studies since 2003 were published), which reflects the increasing significance attached to this issue in nursing clinical practice and research. It underscores the need to work resolutely in reducing VAP and improving QoC for MVPs in critical care settings, particularly in Middle Eastern countries, where six of the studies were conducted. Regarding the geographical distribution of the studies, one study each (7.7%) was conducted in North America and Africa, three (23%) in Europe, and eight (61.5%) in Asia. Evidently there have been few published studies aiming to explore barriers influencing nursesâ success of VAP prevention strategies implementation at Intensive care units.\n\nFurthermore, the revised articles indicate a large degree of methodological homogeneity, with minimal variety in methodological approaches to explore barriers. The majority of the authors in the reviewed studies used quantitative approaches using self-reported questionnaires as the main data collection tool, with the exception of the study by Yeganeh et al. (2019), which used quantitative observational method to determine barriers; and that of Atashi et al. (2018), which used qualitative descriptive study with semi-structured interviews to allow a more in-depth understanding detecting real barriers experienced by nurses in ICUs.\n\nNurses in ICUs experienced various barriers that hinder their ability to implement the recommended VAP measures in a constant and consistent manner (Atashi et al., 2018). From the results of this scoping review, it is apparent that there are many complex, diverse and interconnected barriers that can prevent CCNs from being committed to and compliant with VAP PGs, which accordingly undermines QoC and exposes patients to VAP risk.\n\nThis review showed many barriers within work-environment context playing the paramount role in negatively affecting NC, diverting nurses from being compliant with VAP PGs\n\nWhile medical supplies and equipment in ICU are vital interventional tools for VAP prevention, nurses widely reported a lack of endotracheal tubes with suctioning system and kinetic beds (Aloush et al., 2018; Aloush & Al-Rawajfa, 2020; Jansson et al., 2013; Yeganeh et al., 2019), as well as deficiencies of personal protective equipment like gloves and face masks (Aloush & Al-Rawajfa, 2020), which prevented them from applying appropriate VAP prevention. Yeganeh et al. (2019) observed the unavailability of most essential resources for VAP prevention (including endotracheal tubes with subglottic suctioning, closed suction systems, kinetic beds, etc.), comprising an insurmountable everyday obstacle to nurses that consistently and systematically prevented them from performing important VAP prevention strategies.\n\nAccording to the nurses in Atashi et al. (2018) study, the QoC they delivered to prevent VAP was undermined because they lacked most prerequisite equipment stipulated and necessary in standard guidelines. Moreover, they were unable to prevent VAP because the physical structure of ICU did not include the required materials or permit required activities. For instance, the study reported that there was just one corridor available for the transport of both contaminated and clean materials, suctioning bottles were emptied into toilets, and there were minimal sinks available in the unit for hand washing. The nurses have no control over this problem, because the lack of required resources was due to cost control within the hospital management system. Atashi et al. (2018) concluded that medical supplies and equipment are essential to provide safe and high-QoC for patients, and hospitalsâ cost control policies can negatively influence implementation of guidelines essential for VAP prevention, counter-productively increasing the cost of care over the long term (Al-Sayaghi, 2021).\n\nIn ICU environments, nursing staffing is essential for providing continuous clinical services and improving the QoC of critically ill patients. Staff shortages result in overwork, burnout, and stress among working nurses, reducing nursesâ precision at work and causing the de-prioritization of VAP preventive measures amid a general reduction of QoC (Atashi et al., 2018). There was widespread consensus in the reviewed studies that a lack of staff, which can also be conceptualized as a low nurse-to-patient ratio or high demand, is a common NCB in ICUs at different levels (Al-Sayaghi, 2021; Aloush & Al-Rawajfa, 2020; Atashi et al., 2018; Bankanie et al., 2021; Dehghan et al., 2022; Yaseen & Salameh, 2015).\n\nThe most common barrier to implementing VAP prevention strategies reported by nurses in the studies by Al-Sayaghi (2021) and Dehghan et al. (2022) were the shortage of nursing staff, and these authors argued that inappropriate staff planning is a systemic issue that need to be addressed. Diminution of staff number in the ICU because of cost control policies minimizes the time available for nurses to provide appropriate VAP prevention procedures (to say nothing of holistic and non-biomedical care services, which are utterly disregarded in most busy and under-resourced ICUs). Similarly, Bankanie et al. (2021) concluded that almost all nurses experienced difficulty in adhering with appropriate VAP prevention strategies, despite their knowledge of such guidelines. This indicates that nurses feel overstretched doing many tasks simultaneously, struggling to deliver effective care to patients while juggling numerous patient- and system-related priorities.\n\nThe application of cost control policies results in a reduction of the number of nurses in ICUs, resulting in a reduction in procedure times (Al-Sayaghi, 2021). When nurses work in units with lower bed capacities and lower workloads, they are more likely to comply with policies and guidelines, provide better patient care, and minimize infections (Al-Sayaghi, 2021; Aloush et al., 2018; Aloush & Al-Rawajfa, 2020; Dehghan et al., 2022). This is because appropriate staffing numbers facilitate more time available for the nurses to provide the care required. Time constraints reduce QoC in clinical practice in numerous ways, which in ICUs mainly relate to the absolute prioritization of immediate biomedical needs, and a commensurate de-prioritization of long-term patient needs such as infection prevention and holistic dimensions of care. Many studies indicated that the limited time that nurses have to perform procedures related to VAP prevention is a significant factor in low NC with procedures needed for VAP prevention (Al-Sayaghi, 2021; Hassan & Wahsheh, 2017; Jansson et al., 2013; Kiyoshi-Teo et al., 2014; Ricart et al., 2003; Yaseen & Salameh, 2015).\n\nUnderstaffing in ICUs engenders the lack of time that nurses face, and which contributes to their low NC (Al-Sayaghi, 2021; Yaseen & Salameh, 2015). As a result of cost control policies, the number of nurses in the ICU decreases, making it difficult for procedures to be performed. Guidelines are more likely to be followed by nurses working in units with a lower workload. In an ICU with more nurses, NC would likely be better, and QoC would likely be improved (Al-Sayaghi, 2021). While there was consensus that a lack of time affected the delivery appropriate guidelines for VAP prevention, the reasons to which this barrier were attributed differed among studies. Although common, it was not considered to be one of the main barriers to restrict adherence with guidelines in the study by Yaseen and Salameh (2015). Jansson et al. (2018) noted the very low significance of lack of time on low NC, and reported no effect of staffing levels on NC. Furthermore, a lack of time is not necessarily because of staff shortages. The nature of interventions in ICU also play an important role in maintaining a constant ambience of high-pressure demand and critical workload, which can contribute to the de-prioritization of non-immediate care needs.\n\nHowever, other studies strongly emphasized the role of a lack of time in itself. Kiyoshi-Teo et al. (2014) considered that the time to actually complete the intervention is the only crucial environmental context of NC. Most nurses struggled to finish certain procedures compared to others, and they believed that time availability is positively associated with NC, specifically concerning oral hygiene guidelines. Therefore, time is a limiting factor for NC to complete certain interventions. Similarly, Ricart et al. (2003) reported that some nurses felt overloaded and had no time to perform âhand-washing between patient contactsâ; and Atashi et al. (2018) found that some nurses felt overloaded most of the time, with patient situations necessitating a need for frequent suctioning, rendering it difficult for them to frequently wear gloves for each suctioning, which was flagged as another barrier to effective VAP prevention in ICUs.\n\nMoreover, most nurses in Hassan and Wahsheh (2017) study were overwhelmed with routine ICU procedures due to lack of time, which hindered the proper application of important VAP guideline procedures. While some studies tackle both lack of adequate staffing and lengthy time needed to complete certain procedures, it was not always clear if other reasons were instrumental to the theme of a lack of time. Factors such as ânon-nurse tasksâ and âthe use of electronic documentationâ might be predisposing barriers that contribute to losing time of nurses from performing appropriate VAP prevention performance in ICUs.\n\nIn the included studies of this review, lack of education and training for VAP PGs at hospital settings was brought up many times as NCB in ICUs (Al-Sayaghi, 2021; Aloush et al., 2018; Aloush & Al-Rawajfa, 2020; Atashi et al., 2018; Jansson et al., 2018; Yaseen & Salameh, 2015; Yeganeh et al., 2019). In-service staff education and training respecting VAP prevention can improve nursesâ knowledge and skills and improve care quality. However, most nurses in the above reviewed articles referred to the inadequacy and the ineffectiveness of staff training programs. Many nurses reported that the education they had received in their clinical training was not consistent with the VAP PGs, and more than half of them reported that they never been educated on VAP management courses in their hospitals, and only a third had been educated in the field of mechanical ventilator management based on the VAP PGs (Aloush & Al-Rawajfa, 2020). This finding is congruent with the study of Al-Sayaghi (2021), which reported that a third of nurses never received hospital-based education or training regarding VAP prevention strategies.\n\nYeganeh et al. (2019) observed compiled education and educational seminars about VAP PGs in only a few units, with minimal educational posters and pamphlets. Atashi et al. (2018) revealed that novice nurses did not obtain adequate VAP training, and consequently felt anxious when providing care to their patients after only a week of supervision under senior nurses in their units during their ICU orientation. This ostensibly indicates a lack of necessary basic training for VAP prevention in such hospitals. In addition, the findings of both observation and interviews indicated that most nurses enrolled in VAP PG programs just to get licenses for career development, without really attending or engaging with the learning process wholeheartedly, with a lack of assessment of program effectiveness for improving nursesâ performance (Atashi et al., 2018). This indicates that nurses are less likely to adhere to VAP guidelines because of a lack of training courses and effective, sustainable education programs (Al-Sayaghi, 2021; Aloush et al., 2018; Jansson et al., 2018; Yaseen & Salameh, 2015). Moreover, Aloush et al. (2018) revealed practicing VAP prevention in hospital is not based on research, therefore forming an NCB. Knowledge transfer among nurses is hindered by an inability to translate research into practice (Aloush et al., 2018; Bankanie et al., 2021), and poor information-sharing between them, which is reflected in poor education and continuing professional education among nurses, associated with low employee satisfaction and QoC delivery (Bankanie et al., 2021).\n\nAvailability of written policies and protocol of nursing care for VAP prevention for MVP is essential and effective in ICUs to enhance nursesâ adherence with recommended guidelines. Studies found that more than half of the nurses deemed a lack of VAP prevention policy and protocol in their facilities to be a major NCB (Aloush & Al-Rawajfa, 2020; Atashi et al., 2018; Hassan & Wahsheh, 2017; Yaseen & Salameh, 2015). Moreover, nurses in Atashi et al.âs (2018) study argued that irrelevant VAP prevention protocols and recommendations established in foreign countries are being applied in settings where they are not necessarily applicable, and they recommended developing new institutionally sensitive guidelines, considering the features of particular settings, such as the number of nurses in shifts, specifications, and equipment availability. On the other hand, Aloush et al. (2018) found that nursing practice in ICUs not being based on research findings (i.e., a lack of evidence-based practice) is an NCB, reflected in nursesâ lack of awareness of updated knowledge for VAP prevention. Therefore, action is needed by healthcare systems and nurse educators to impart up-to-date evidence-based care, particularly educational sessions to explain protocols and policies for VAP prevention based on evidence-based sources, to help familiarize and orient nurses in order to expedite application of guidelines in practice contexts.\n\nIn addition, healthcare systems need to assess and improve the knowledge and capabilities of nurse educators and senior nurses to enable the diffusion of required VAP prevention knowledge and practice, aside from making training and educational materials available. The supervision of nurses in ICUs regarding their skills is a significant component behind improvement of nurses performance, and a lack of supervision is a barrier commonly identified by nurses themselves (Atashi et al., 2018), and the related issue of the absence of guidance for achieving standard VAP prevention, which is another known NCB (Aloush et al., 2018; Jansson et al., 2013). Such features could be due to related barriers such as lack of time for monitoring, lack of supervision-related training, and lack of knowledge and abilities for efficient supervision by senior nurses who can guide practice.\n\nAtashi et al. (2018) identified ineffective supervision as a main barrier for effective VAP prevention. Supervisors need to perform many nonsupervisory duties, which prevent them from effectively performing their activities related to supervision. For instance, they were mostly involved in resolving interpersonal conflicts, and making necessary adjustments for patient transfers to other hospitals. Managing educational resources involves the coordination of human and material resources to monitor, plan, strategize, and implement the delivery of education. Based on the reviewed articles, inadequate and inefficient staff training and education at clinical settings suggest the need of hospitals to adhere with the CDCâs 2003 recommendations on the importance of conducting education and training programs of nurses in ICUs (Tablan et al., 2003), for effective implementation of VAP prevention strategies.\n\nRelated to the resource constraints described previously, which are related to hospital management, managementâs direct support for senior nurses, including managers and supervisors, is also essential for the success of VAP implantation. Four reviewed studies found that insufficient management support is an NCB (Aloush et al., 2018; Atashi et al., 2018; Bankanie et al., 2021; Dehghan et al., 2022; Jansson et al., 2018). Passive and ineffective management affects the accuracy in task performance of nurses, forming an NCB (Atashi et al., 2018). Other barriers than nurses believed as barriers include limited professional competence, low job motivation, and limited professional accountability, all of which reflect organizational and systemic issues. Nursing managers need to understand how the scarcity of hierarchical support and passive human resource management have impacts on the implementation success of guidelines.\n\nJansson et al. (2018) reported that passive management was instrumental in nursesâ ambiguous perceptions of their role in VAP prevention strategies. Role ambiguity was identified as a barrier because nurses are uncertain about their definite tasks in implementing VAP prevention. A lack of clarity about expected roles may cause nurses to struggle, despite their intrinsic role in the evidence-based practice paradigm, the policies of individual hospitals, and of health systems in general, may not be clear for nurses (e.g., whether sedation interruption is a nursing task, or whether only physicians have this authority). Hospital management needs to provide more support by clarifying and setting clear rules and responsibilities for nurses in implementing strategies.\n\nMoreover most of the nurses in Bankanie et al.âs (2021) study noted low âjob discretionâ to be a barrier. Nurses are not allowed to make responsible choices, judgments, or decisions with their patients in ICU. This might be as a result of unsupportive system along with job ambiguity in addition to lack of knowledge or skills. Nurses in the study of Dehghan et al. (2022) indicated that a lack of managerial support within the hospitals greatly undermines NC, which is related directly to healthcare organizational systems. These barriers reflect the low quality of working life, and poor organizational culture, which directly influence nursesâ satisfaction and their readiness to deliver higher QoC.\n\nThe work environment also includes social features within team that can impact workplace relationships, collaboration, efficiency at work, which have impacts on NC. Nurses are the most vital members within healthcare teams, and their role as patient advocates and holistic care specialists is paramount in managing VAP impacts on ICU patients. In this role, nurses must work with each other as peers, and with other healthcare professions such as intensivists and respirologists, to prevent VAP in ICUs. Based on the findings within the review, nurses may be affected by peer influence and teamwork issues.\n\nIssues of peers and teams significantly influence nursing performance, and adherence with guidelines, which also intersects with the issue of role models; nurses revealed that the lack of professional role models during their working activities comprises an NCB (Aloush et al., 2018). Nurses mentioned that the lack of a team-based approach to care and interventions is a big NCB, which might be due to severe shortages (Dehghan et al., 2022). In their study, Atashi et al. (2018) found that colleaguesâ negative professional attitudes inhibit accurate VAP prevention practice. Participantsâ reports indicated that some colleagues failed to perform their work accurately. They sometimes documented that they performed procedures for VAP prevention when they really did not, such as documenting endotracheal tube cuff pressure without actually measuring it.\n\nNurse-related barriers included issues related to limited personal competences (e.g., education and knowledge, skills, and experience), and situation-related barriers (e.g., concerning the ICU clinical context), which pertain to the essential prerequisites for the success of VAP prevention in ICUs.\n\nLimited personal competencies\n\nBased on the reviewed articles, personal characteristics may be instrumental in NC. The findings showed a lack of NC might be entrenched in nursing education. Aloush et al. (2018) reported that 63% of nurses in their study had received no education about VAP PGs in their nursing education, and the low level of VAP-related education among ICU nurses directly undermined their NC. They also revealed that there was a statistically significant difference based on academic degree, whereby nurses with mastersâ degrees had higher NC in comparison with those with baccalaureate and diploma degrees, which was later affirmed by Bankanie et al. (2021). Aloush and Al-Rawajfa (2020) reported that the education nurses received during their study was not consistent with VAP PGs, adding a further barrier to their NC. It is important for schools of nursing to consider improving educational programs to improve nursesâ ICU knowledge and VAP-related education, consistent with clinical guidelines, to subsequently enhance nursesâ NC and QoC when they transition to clinical practice (Aloush et al., 2018; Aloush & Al-Rawajfa, 2020; Yaseen & Salameh, 2015).\n\nSome studies reported a lack of skills to be an NCB. Jansson et al. (2013) and Bankanie et al. (2021) stated that a lack of skills among nurses was a significant barrier to compliance with guidelines for VAP prevention, and they called for ongoing educational interventions and effective strategies to facilitate knowledge and skills dissemination and transfer in the workplace. Almost a quarter (23%) of nurses âstrongly disagreedâ that a lack of skills was considered a barrier in a study by Aloush and Al-Rawajfa (2020), but this may reflect social desirability bias (i.e., nurses not wishing to acknowledge to others or to themselves that they lacked the required skills for serious interventions due to perceiving this to be a personal rather than systemic and educational shortcoming).\n\nAloush et al. (2018) reported that years of experience was a significant indicator for increased NC, and numerous other studies found that less experienced nurses had lower NC in Middle Eastern contexts (Aloush & Al-Rawajfa, 2020; Yaseen & Salameh, 2015); conversely, Jansson et al. (2013, 2018) found no relation between NC and longer work experience in Finland.\n\nSome nurses questioned the importance of VAP prevention measures to patient well-being because, in certain clinical situations, they were required to make appropriate judgments and timely decisions when encountering patients with serious physiological issues. The reason for these concerns could be that certain clinical guidelines do not adequately consider individual patient needs and capabilities (Jansson et al., 2018). Unpredictable adverse effects harming patients, and undesirable patient outcomes from some VAP preventive procedures, were cited as dreaded outcomes by nurses in many studies (Al-Sayaghi, 2021; Dehghan et al., 2022; Hassan & Wahsheh, 2017; Jansson et al., 2018; Ricart et al., 2003).\n\nJansson et al. (2018) reported that nursesâ concerns regarding the impacts of VAP prevention procedures may have hindered adherence with appropriate strategies. In particular, nurses were doubtful of the indications of sedation, and worried about over-sedation that might harm patients. They also were worried about potential mistakes due to keeping their patients in a semi-recumbent position, as it is difficult to estimate the appropriate angle of the head of the bed. Uncertainty about indications of enhanced oral care, the estimated depth during endotracheal suctioning, and the duration of suctioning added to their fear of potential complications. Other studies reported that nurses were commonly concerned about the detachment of attached tubes during certain VAP prevention procedures (Dehghan et al., 2022). These concerns might be because of the fear of committing mistakes per se; producing undesired alterations in hemodynamic status of patients; or the belief that performing VAP measures could cause deterioration in the critical status of patients. These concerns made the nurses more cognizant of their patientsâ holistic needs, and they experienced a dilemma between the biomedical mandates of VAP prevention and what they perceived to be their nursing duty of safeguarding patients holistically, which resulted in noncompliance with some VAP preventive measures.\n\n\nConclusion\n\nCommon barriers appear to inhibit nurses from performing appropriate VAP prevention strategies. This review highlighted the intricate correlated barriers that inhibit NC with evidence-based VAP prevention strategies. The aim of this review to identify gap in literature to guide future research. The ICU work environment and hospital management play major roles in creating low NC, without appropriate measures to address nursing and situational impediments to compliance, such as the effects of attitudes and behaviors, efficacy, low job motivation, peer influence and team dynamics, all of which need further scrutiny and clarification. Contextual and work environment barriers are relatively under-reported, and warrant further exploration, but it is clear that the work environment is the base issue that triggers multiple NCBs, beyond the control of individual nurses.\n\nMoreover, future use of different methodological approaches, such as mixed-methods studies and more exploratory qualitative studies, can gain more in-depth insights, and find out the thorough predisposing barriers formulating a solid literature. Mixed-method designs might be beneficial to identify factors that affected NC. For instance, an observational study followed by qualitative study, or conducting a qualitative study to discover the barriers, followed by administration of a self-reported survey created based on the qualitative findings, would provide a robust research approach. Comparative studies of hospitals with zero VAP rate and high VAP rate within same and/or other country are essential to solidly elucidate real barriers. Follow-up studies to identify the barriers, plan and then implement changes to improve NC and monitor related KPIs (particularly VAP rate) would clarify systemic and long-term barriers more fully. Such future research may help to solidly elucidate all barriers that might be essential for nurse leaders and policy makers, particularly if various types of study are conducted within the same local context.\n\nThis scoping review can be used as a template for future studies, representing the key concepts underpinning NCBs among nurses in intensive care units according to current evidence. Further primary research about barriers would be beneficial to support nursing leaders and healthcare systems in augmenting compliance and informing practice, to positively influence QoC for MVPs, empowering nurses to able to identify and control their own barriers.\n\nThe duration of the search encompassed works published since 2003, which was included all recent works published in English. The numerous specializations involved in intensive care units allow generalization of the findings of this study for all CCNs, in terms of barriers hindering nursing compliance with VAP prevention strategies. Moreover, this is the first scoping review that considered a standalone study for future research using a rigorous PRISMA-ScR report, adding more strength to the review.\n\nHowever, this scoping review also has some limitations. To make it more feasible, this review included only published, peer reviewed research articles in English language, available in full-text form. These criteria may have led to missing some relevant studies and information. Future reviews may further include other types of literature, such as grey literature, reports, dissertations, and editorials, and works in non-English languages. Future reviews in this topic may compare published findings with those of unpublished literature.",
"appendix": "Data availability\n\nFigshare: Barriers to compliance with evidence-based guidelines for ventilator-associated pneumonia among critical care nurses: A scoping review (Al-Tamimi et al., 2022). https://doi.org/10.6084/m9.figshare.21493122\n\nThe project contains the following underlying data:\n\nSearch Strategy. File 1- PubMed search strategy performed June 13, 2022\n\nRepository name: Figshare: https://doi.org/10.6084/m9.figshare.21493122\n\nThis project contains the following extended data:\n\n⢠Data extraction form. Summary review of included studies)\n\nFigshare: PRISMA-Scr checklist for âBarriers to compliance with evidence-based guidelines for ventilator-associated pneumonia among critical care nurses: A scoping reviewâ. https://doi.org/10.6084/m9.figshare.21493122\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAl-Sayaghi KM: Critical care nursesâ compliance and barriers toward ventilator-associated pneumonia prevention guidelines: Cross-sectional survey. Journal of Taibah University Medical Sciences. 2021; 16(2): 274â282. PubMed Abstract | Publisher Full Text\n\nAl-Tamimi M, Ahmed FR, Bani-Issa W:Barriers to compliance with evidence-based guidelines for ventilator-associated pneumonia among critical care nurses: A scoping review. [DATASET]. figshare. 2022. Publisher Full Text\n\nAloush SM, Abdelkader FA, Al-Sayaghi K, et al.: Compliance of nurses and hospitals with ventilator-associated pneumonia prevention guidelines: A Middle Eastern survey. J. Nurs. Care Qual. 2018; 33(3): E8âE14. PubMed Abstract | Publisher Full Text\n\nAloush SM, Al-Rawajfa OM: Prevention of ventilator-associated pneumonia in intensive care units: Barriers and compliance. Int. J. Nurs. Pract. 2020; 26(5): e12838. Article e12838. PubMed Abstract | Publisher Full Text\n\nAmerican Thoracic Society, & Infectious Diseases Society of America: Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am. J. Respir. Crit. Care Med. 2005; 171(4): 388â416. Publisher Full Text\n\nAtashi V, Yousefi H, Mahjobipoor H, et al.: The barriers to the prevention of ventilator-associated pneumonia from the perspective of critical care nurses: A qualitative descriptive study. J. Clin. Nurs. 2018; 27(5â6): e1161âe1170. PubMed Abstract | Publisher Full Text\n\nBankanie V, Outwater AH, Wan L, et al.: Assessment of knowledge and compliance to evidence-based guidelines for VAP prevention among ICU nurses in Tanzania. BMC Nurs. 2021; 20(1): 209. Article 209. PubMed Abstract | Publisher Full Text\n\nBeattie M, Shepherd A, Maher S, et al.: Continual improvement in ventilator acquired pneumonia bundle compliance: A retrospective case matched review. Intensive Crit. Care Nurs. 2012; 28(5): 255â262. Publisher Full Text\n\nBird D, Zambuto A, OâDonnell C, et al.: Adherence to ventilator-associated pneumonia bundle and incidence of ventilator-associated pneumonia in the surgical intensive care unit. Arch. Surg. 2010; 145(5): 465â470. PubMed Abstract | Publisher Full Text\n\nCason CL, Tyner T, Saunders S, et al.: Nursesâ implementation of guidelines for ventilator-associated pneumonia from the Centers for Disease Control and Prevention. Am. J. Crit. Care. 2007; 16(1): 28â37. PubMed Abstract | Publisher Full Text\n\nCrunden E, Boyce C, Woodman H, et al.: An evaluation of the impact of the ventilator care bundle. Nurs. Crit. Care. 2005; 10(5): 242â246. Publisher Full Text\n\nda Cruz JRM , da Silva Martins MD : Pneumonia associated with invasive mechanical ventilation: Nursing care. Revista de Enfermagem Referência. 2019; IV Série(20): 87â96. Publisher Full Text\n\nDehghan M, Arab M, Akafzadeh T, et al.: Intensive care unit registered nursesâ perceived barriers towards ventilated associated pneumonia prevention in southeast Iran: A cross-sectional descriptiveâanalytical study. BMJ Open. 2022; 12(9): e064147. Article e064147. PubMed Abstract | Publisher Full Text\n\nEl-Saed A, Al-Jardani A, Althaqafi A, et al.: Ventilator-associated pneumonia rates in critical care units in 3 Arabian Gulf countries: A 6-year surveillance study. Am. J. Infect. Control. 2016; 44(7): 794â798. PubMed Abstract | Publisher Full Text\n\nElo S, KyngÀs H: The qualitative content analysis process. J. Adv. Nurs. 2008; 62(1): 107â115. Publisher Full Text\n\nHassan ZM, Wahsheh MA: Knowledge level of nurses in Jordan on ventilator-associated pneumonia and preventive measures. Nurs. Crit. Care. 2017; 22(3): 125â132. PubMed Abstract | Publisher Full Text\n\nHunter JD: Ventilator associated pneumonia. BMJ (Clinical Research Ed.). 2012; 344. Article e3325. Publisher Full Text\n\nIbrahim EH, Tracy L, Hill C, et al.: The occurrence of ventilator-associated pneumonia in a community hospital: Risk factors and clinical outcomes. Chest. 2001; 120(2): 555â561. PubMed Abstract | Publisher Full Text\n\nInstitute for Healthcare Improvement: How-to guide: Prevent ventilator-associated pneumonia. Institute for Healthcare Improvement;2012.\n\nJahansefat L, Vardanjani MM, Bigdelian H, et al.: Exploration of knowledge of, adherence to, attitude and barriers toward evidence-based guidelines (EBGs) for prevention of ventilator-associated pneumonia (VAP) in healthcare workers of pediatric cardiac intensive care units (PCICUs): A quali-quantitative survey. International Journal of Medical Research & Health Sciences. 2016; 5(9): 67â73.\n\nJam R, Mesquida J, Hernández Ã, et al.: Nursing workload and compliance with non-pharmacological measures to prevent ventilator-associated pneumonia: A multicentre study. Nurs. Crit. Care. 2018; 23(6): 291â298. PubMed Abstract | Publisher Full Text\n\nJansson M, Ala-Kokko T, Ylipalosaari P, et al.: Critical care nursesâ knowledge of, adherence to and barriers towards evidence-based guidelines for the prevention of ventilator-associated pneumonia - A survey study. Intensive Crit. Care Nurs. 2013; 29(4): 216â227. PubMed Abstract | Publisher Full Text\n\nJansson MM, SyrjÀlÀ HP, Talman K, et al.: Critical care nursesâ knowledge of, adherence to, and barriers toward institution-specific ventilator bundle. Am. J. Infect. Control. 2018; 46(9): 1051â1056. PubMed Abstract | Publisher Full Text\n\nKiyoshi-Teo H, Cabana MD, Froelicher ES, et al.: Adherence to institution-specific ventilator-associated pneumonia prevention guidelines. Am. J. Crit. Care. 2014; 23(3): 201â215. PubMed Abstract | Publisher Full Text\n\nKlompas M: What is new in the prevention of nosocomial pneumonia in the ICU? Curr. Opin. Crit. Care. 2017; 23(5): 378â384. Publisher Full Text\n\nKlompas M, Branson R, Cawcutt K, et al.: Strategies to prevent ventilator-associated pneumonia, ventilator-associated events, and nonventilator hospital-acquired pneumonia in acute-care hospitals: 2022 update. Infect. Control Hosp. Epidemiol. 2022; 43(6): 687â713. PubMed Abstract | Publisher Full Text\n\nMagill SS, O'Leary E, Janelle SJ, et al.: Changes in prevalence of health care-associated infections in U.S. hospitals. N. Engl. J. Med. 2018; 379(18): 1732â1744. PubMed Abstract | Publisher Full Text\n\nMetersky ML, Kalil AC: Management of ventilator-associated pneumonia: Guidelines. Clin. Chest Med. 2018; 39(4): 797â808. Publisher Full Text\n\nOsti C, Wosti D, Pandey B, et al.: Ventilator-associated pneumonia and role of nurses in its prevention. Journal of the Nepal Medical Association. 2017; 56(208): 461â468. PubMed Abstract | Publisher Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: An updated guideline for reporting systematic reviews. BMJ (Clinical Research Ed.). 2021; 372. Article n71. PubMed Abstract | Publisher Full Text\n\nPapazian L, Klompas M, Luyt CE: Ventilator-associated pneumonia in adults: A narrative review. Intensive Care Med. 2020; 46(5): 888â906. PubMed Abstract | Publisher Full Text\n\nPeters MD, Godfrey CM, Khalil H, et al.: Guidance for conducting systematic scoping reviews. JBI Evidence Implementation. 2015; 13(3): 141â146. Publisher Full Text\n\nPeters MD, Marnie C, Tricco AC, et al.: Updated methodological guidance for the conduct of scoping reviews. JBI Evidence Synthesis. 2020; 18(10): 2119â2126. PubMed Abstract | Publisher Full Text\n\nPogorzelska M, Stone PW, Furuya EY, et al.: Impact of the ventilator bundle on ventilator-associated pneumonia in intensive care unit. Int. J. Qual. Health Care. 2011; 23(5): 538â544. PubMed Abstract | Publisher Full Text\n\nRello J, Afonso E, Lisboa T, et al.: A care bundle approach for prevention of ventilator-associated pneumonia. Clin. Microbiol. Infect. 2013; 19(4): 363â369. Publisher Full Text\n\nRello J, Lorente C, Bodà M, et al.: Why do physicians not follow evidence-based guidelines for preventing ventilator-associated pneumonia?: A survey based on the opinions of an international panel of intensivists. Chest. 2002; 122(2): 656â661. Publisher Full Text\n\nRicart M, Lorente C, Diaz E, et al.: Nursing adherence with evidence-based guidelines for preventing ventilator-associated pneumonia. Crit. Care Med. 2003; 31(11): 2693â2696. PubMed Abstract | Publisher Full Text\n\nSafdar N, Dezfulian C, Collard HR, et al.: Clinical and economic consequences of ventilator-associated pneumonia: A systematic review. Crit. Care Med. 2005; 33(10): 2184â2193. PubMed Abstract | Publisher Full Text\n\nTabaeian S, Yazdannik A, Abbasi S: Compliance with the standards for prevention of ventilator-associated pneumonia by nurses in the intensive care units. Iran. J. Nurs. Midwifery Res. 2017; 22(1): 31â36. PubMed Abstract | Publisher Full Text\n\nTablan OC, Anderson LJ, Besser RE, et al.: Guidelines for preventing health-care-associated pneumonia, 2003: Recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee. 2003. (Report No. 53(RR03);1-36). CDC Morbidity and Mortality Weekly Report.Reference Source\n\nTorres A, Niederman MS, Chastre J, et al.: International ERS/ESICM/ESCMID/ALAT guidelines for the management of hospital-acquired pneumonia and ventilator-associated pneumonia: Guidelines for the management of hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) of the European Respiratory Society (ERS), European Society of Intensive Care Medicine (ESICM), European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and Asociación Latinoamericana del Tórax (ALAT). Eur. Respir. J. 2017; 50(3). Article 1700582. PubMed Abstract | Publisher Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and explanation. Ann. Intern. Med. 2018; 169(7): 467â473. PubMed Abstract | Publisher Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: A scoping review on the conduct and reporting of scoping reviews. BMC Med. Res. Methodol. 2016; 16: 15. Article 15. PubMed Abstract | Publisher Full Text\n\nValles J, Artigas A, Rello J, et al.: Continuous aspiration of subglottic secretions in preventing ventilator-associated pneumonia. Ann. Intern. Med. 1995; 122(3): 179â186. PubMed Abstract | Publisher Full Text\n\nWarren DK, Shukla SJ, Olsen MA, et al.: Outcome and attributable cost of ventilator-associated pneumonia among intensive care unit patients in a suburban medical center. Crit. Care Med. 2003; 31(5): 1312â1317. PubMed Abstract | Publisher Full Text\n\nYaseen RW, Salameh TN: Saudi critical care nurses' knowledge of and barriers toward adherence to prevention of ventilator associated pneumonia guidelines. Journal of Nursing and Health Science. 2015; 4(2): 65â69. Publisher Full Text\n\nYeganeh M, Yekta H, Farmanbar R, et al.: Knowledge of evidence-based guidelines in ventilator-associated pneumonia prevention. J. Evid. Based Med. 2019; 12(1): 16â21. PubMed Abstract | Publisher Full Text\n\nZimlichman E, Henderson D, Tamir O, et al.: Health careâassociated infections: A meta-analysis of costs and financial impact on the US health care system. JAMA Intern. Med. 2013; 173(22): 2039â2046. Publisher Full Text"
}
|
[
{
"id": "158512",
"date": "16 Jan 2023",
"name": "Khaled Mohammed Al-Sayaghi",
"expertise": [
"Reviewer Expertise Critical Care Nursing",
"infection control practices",
"nutritional support in critical care",
"and mechanical ventilation"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall the review is so good. It provides information about the barriers to compliance with evidence-based guidelines for VAP among critical care nurses. It considered one of the important topics in the critical care nursing. I thank the authors for choosing this important topic. Kindly consider the following comments:\nThe small paragraphs in the introduction can be merged into 4 large paragraphs.\n\nHas the systematic review been registered in specific institutions such as PROSPERO?\n\nWhen conducting a systematic review, it is normal to provide some additional information on the search methodology (search strategy).\n\nRegarding the search strategy, I see that synonyms of the keywords for the research topic can be helpful. Synonyms provide access to all the research published on the subject of the research, for example, barriers may use factors, nurse may use health care workers, guideline may use bundle, compliance may use adherence, intensive care may use critical care, etc. The use of the single term is likely to miss quite a lot of the literature on this subject.\n\nIs it possible to pool the effect size of each compliance barriers among critical care nurses by meta-analysis? If not, please explain the reasons.\n\nThe researchers in the discussion did not talk about the secondary research that dealt with the subject in some detail and how this study differs from previous reviews.\n\nYou need to provide operational definitions for each barrier you mentioned in the results.\n\nThe work environment-related barriers were categorized into several subcategories. If possible, categorize the âNurse-related barriersâ and âSituation-related barriersâ.\n\nPage 13, line 11: âLimited personal competenciesâ is the only subcategory under the âNurse-related barriersâ. If it is not possible to add other subcategories, it is better to remove the subtitle.\n\nPage 14: there is a redundancy in the second and third paragraphs of conclusions. It needs to be summarized in one small paragraph.\n\nYou need to add a subheading about implications for nursing.\n\nThe paper needs professional English editing.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "9472",
"date": "12 Apr 2023",
"name": "Muna Al-Tamimi",
"role": "Author Response",
"response": "Dear Dr. al-Sayaghi, Thank you for facilitating the peer review. The revised version of the scoping review has been uploaded, and we have addressed all of Dr. al-Sayaghi's comments to further improve the quality of our paper. In response to each comment, we have provided the following responses: 1. The small paragraphs in the introduction can be merged into 4 large paragraphs. We have merged the paragraphs as the following: Changes within the new version. Nurses play a pivotal role in providing safe and direct care to MVPs, including practicing VAP prevention strategies that are essential to prevent infection and therefore improve the quality of patient care and outcomes in ICUs (Jansson et al., 2013; Osti et al., 2017) According to Pogorzelska et al. (2011), nursing compliance with VAP prevention guidelines in ICUs (hereinafter âNCâ) should be at least 95%, in order to effectively reduce VAP incidence. Therefore, strict compliance with strategies and recommendations is required from nurses, who must appropriately perform several interventions and procedures to achieve optimum outcomes (Miranda da Cruz & da Silva Martins, 2019; Rello et al., 2013; Tabaeian et al., 2017). Facilitating and promoting changes in patient care, and encouraging NC are crucial for VAP prevention success, and also providing regular feedback on process measure performance and outcome rates are among the best practices to facilitate adherence to guidelines (Crunden et al., 2005; Klompas, 2017). In spite of the existence of clinical practice guidelines for preventing VAP, these guidelines are not consistently followed (Cason et al., 2007; Ricart et al., 2003). Reported levels of compliance with and proper use of strategies vary widely in various contexts, across health systems, specialties, and nurses, ranging from 20% to nearly 100% (Beattie et al., 2012; Bird et al., 2010; Rello et al., 2002). Several studies showed that nurses generally exhibit low NC, which could be attributable to diverse factors (Aloush et al., 2018; Aloush & Al-Rawajfa, 2020;Jahansefat et al., 2016;Jam et al., 2018). Given the critical impact of NC on patientsâ QoC, it is essential to identify the factors influencing it that might impede the proper implementation of EBGs, and hence affect patient outcomes.  2. Has the systematic review been registered in specific institutions such as PROSPERO This scoping review follows Joanna Briggs Institute (JBI) Manual for Evidence Synthesis. Available in https://jbi-global-wiki.refined.site/space/MANUAL/4687810/11.2+Development+of+a+scoping+review+protocol Therefore, it was not registered in PROSPERO, however, this scoping review is registered with Figshare ( https://figshare.com) as following details: Data availability: Underlying data Al-Tamimi et al., 2022 Figshare: Barriers to compliance with evidence-based guidelines for ventilator-associated pneumonia among critical care nurses: A scoping review. https://doi.org/10.6084/m9.figshare.21493122 The project contains the following underlying data: Search Strategy. File 1- PubMed search strategy performed June 13, 2022 Extended data Repository name: Figshare: https://doi.org/10.6084/m9.figshare.21493122 This project contains the following extended data: Data extraction form. Summary review of included studies. Reporting guidelines Figshare: PRISMA-Scr checklist for âBarriers to compliance with evidence-based guidelines for ventilator-associated pneumonia among critical care nurses: A scoping reviewâ. https://doi.org/10.6084/m9.figshare.21493122 Data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication). 3. When conducting a systematic review, it is normal to provide some additional information on the search methodology (search strategy). The detailed PubMed search strategy is available as an additional file in the data availability \"Underlying dataâ. Search Strategy. File 1- PubMed search strategy performed June 13, 2022 It can also be accessed through the extended data: Repository name: Figshare: https://doi.org/10.6084/m9.figshare.21493122 4. Regarding the search strategy, I see that synonyms of the keywords for the research topic can be helpful. Synonyms provide access to all the research published on the subject of the research, for example, barriers may use factors, nurse may use health care workers, a guideline may use bundle, compliance may use adherence, intensive care may use critical care, etc. The use of the single term is likely to miss quite a lot of the literature on this subject. Following PRISMA-ScR checklist, we added one full electronic search strategy of Pubmed. This information is available in F1000Research Search Strategy. File 1- PubMed search strategy performed June 13, 2022. And in the Extended data Repository name: Figshare: https://doi.org/10.6084/m9.figshare.21493122. However, search strategies of the other databases (scopus and CINHAL) are available upon request that includes other synonyms. Therefore, the following sentence is added to clarify this point.  âAdditional search strategies are available upon request from the corresponding authorâ. 5. Is it possible to pool the effect size of each compliance barriers among critical care nurses by meta-analysis? If not, please explain the reasons. As per the followed JBI guidelines, meta-analysis was not conducted for the scoping review paper. https://jbi-global-wiki.refined.site/space/MANUAL/4687681/11.2.8+Analysis+of+the+evidence 6. The researchers in the discussion did not talk about the secondary research that dealt with the subject in some detail and how this study differs from previous reviews. This first scoping review is considered a standalone study for future research using a rigorous PRISMA-ScR report. It includes peer-reviewed internationally published articles that mainly focus on barriers toward compliance with evidence-based guidelines for ventilator-associated pneumonia among critical care nurses. It aims to examine the characteristics of studies, identify, and summarize key findings of related studies; and identify gaps in extant studies that may help inform future research in this area. These aims are prominent for any scoping review paper. 7. You need to provide operational definitions for each barrier you mentioned in the results. The comment is taken into consideration by adding the following operational definitions for each barrier. Changes within the new version. Work environment-related barriers: Barriers related to the work environment are those beyond the control of individual nurses (Jansson et al. 2018). These include significant challenges in the workforce that hinder nurses from complying with VAP prevention EBGs. Nurse-related barriers: These barriers are nurse-related characteristics that limit the nurse's ability to follow the guidelines (Aloush et al., 2018; Yaseen & Salameh, 2015). Situation-related barriers are factors related to patients that hinder nursesâ compliance toward VAP guidelines (Jansson et al., 2018; Ricart et al., 2003) and the doubts that can influence nursesâ behavior toward these guidelines (Dehghan et al., 2022; Jansson et al., 2018). 8. Work environment-related barriers were categorized into several subcategories. If possible, categorize the âNurse-related barriersâ and âSituation-related barriers Based on the revised articles and synthesis of results, further categorization to the âNurse-related barriersâ and âSituation-related barriers was not viable. 9. Page 13, line 11: âLimited personal competenciesâ is the only subcategory under the âNurse-related barriersâ. If it is not possible to add other subcategories, it is better to remove the subtitle. The comment is considered. subtitle removed. 10. Page 14: there is a redundancy in the second and third paragraphs of conclusions. It needs to be summarized in one small paragraph.   A summary of the conclusion is conducted as per the recommendation as follows; This scoping review can be used as a template for future studies, representing the key concepts underpinning NCBs among nurses in intensive care units. Further primary research about barriers using different methodological approaches, such as mixed-methods studies and more exploratory qualitative studies can gain more in-depth insights and find out the thorough predisposing barriers formulating a solid literature. Comparative studies of hospitals with zero VAP rate and high VAP rate within the same and/or other countries are essential to solidly elucidate real barriers. Follow-up studies to identify the barriers, plan and then implement changes to improve NC, and monitor related KPIs (particularly VAP rate) would clarify systemic and long-term barriers more fully. Such research may help to solidly elucidate all barriers that might be essential for nurse leaders, and policymakers particularly if various types of study are conducted within the same local context. It would be beneficial to support nursing leaders and healthcare systems in augmenting compliance and informing practice, positively influencing QoC for MVPs, and empowering nurses to able to identify and control their own barriers. 11. You need to add a subheading about implications for nursing. Implications were included in the conclusion (as per PRISMA-ScR checklist). 12. The paper needs professional English editing.          The paper has been professionally edited by a native speaker (UK)."
}
]
},
{
"id": "158511",
"date": "16 Jan 2023",
"name": "Intima Alrimawi",
"expertise": [
"Reviewer Expertise Her research focuses on improving the quality of care for vulnerable families and children with complex health concerns and critical illness and developing nursing education",
"mainly in low and middle-income countries."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis scoping review discusses the topic of barriers to compliance with evidence-based guidelines for ventilator-associated pneumonia among critical care nurses. This is a very important topic that was not covered in other scoping reviews.\n\nIn the methodology section the researcher thoroughly discussed the scientific approach that they followed to conduct this review.\n\nThe results section needs to be developed further; additional discussion is needed around the selected studies and the final themes that did emerge from them.\nThe discussion section needs to take the themes to the next level and discuss the potential implication and recommendations to recommend these barriers. For example, if you think that lack of staff and time was a barrier, then what is the implication of this in the current practice, and how can we overcome this barrier on the ground?\n\nThe conclusion section is long, it needs to be summarized and be more focused.\nYou can also add a separate recommendation section.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/11-1551
|
https://f1000research.com/articles/10-1027/v1
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11 Oct 21
|
{
"type": "Research Article",
"title": "Essential oil of Cymbopogon martini, source of geraniol, as a potential antibacterial agent against Bacillus subtilis, a pathogen of the bakery industry",
"authors": [
"Sara Santamarta",
"A. Cristina Aldavero",
"M Angeles Rojo",
"Sara Santamarta",
"A. Cristina Aldavero"
],
"abstract": "Background: Bacteria can adhere and grow on any surface due to their chemical and physical interaction, leading to the development of biofilms. Essential oils have a great potential for use in the food industry, as they can effectively prevent the presence of some pathogenic microorganisms. Species such as those in the Bacillus genus have the ability to produce toxins. Some strains of Bacillus subtilis have been related to cases of foodâborne diseases. In the bakery industry, B. subtilis also has been related to âropeâ disease, linked to bread preservation processes. Methods: The aim of the study was to analyse the antibacterial properties of 24 chemotyped essential oils against the growth of B. subtilis. The biological activity study was carried out using disk diffusion in agar and broth dilution methods. Results: The essential oil of Cymbopogon martinii var. motia had a high geraniol content (>80.53%) and showed a high antimicrobial effect against the Gram-positive bacterium B. subtilis. Binary combinations of Cymbopogon martinii var. motia oil with Eugenia caryophyllus showed antagonistic effects on B. subtilis. Conclusions:  The essential oil of Cymbopogon martinii var. motia has an interesting potential use in the bakery industry as a preservative, in applications such as nano encapsulation for bakery doughs, active packaging of baked products, or surface disinfectants.",
"keywords": [
"Bacillus subtillis",
"Cymbopogon martinii",
"antibacterial activity",
"geraniol",
"rope disease",
"bakery industry"
],
"content": "Introduction\n\nEssential oils (EOs) are aromatic and volatile natural compounds, synthesized and secreted by specialized histological structures. They are extracted from plant material, such as flowers, aerial parts, roots, bark, leaves and fruits.1 EOs are secondary metabolites playing a role in the plant protection against biotic and abiotic stress. They constitute about 1% of plant secondary metabolites.2 The composition of EOs from the same species of plant can vary with geographic location, the harvesting season, the part of the plant being distilled, or extraction method.3,4\n\nThe chemical composition of EOs is complex; some of them have around 20 to 60 different bioactive components, only two or three of which are in concentrations within a range of 20% to 70%, with the rest found in trace amounts. Within this mix of bioactive components, two different compound groups can be identified: terpenes and terpenoids. Terpenes are the most abundant components in EOs, and are classified into monoterpenes and sesquiterpenes, according to their number of isoprene units. Within this group, α-pinene, β-caryophyllene, γ-terpinene, limonene, geraniol, and p-cymene are included. Monoterpenes can be linear (acyclic) or contain rings (monocyclic and bicyclic). Modified terpenes (containing oxygen molecules or lacking a methyl group) are called monoterpenoids, and include carvacrol, thymol, menthol, borneol, geranyl acetate, 1,8-cineole, and linalool. Terpenoids, present in the EOs (less predominant) are the aromatic compounds, derivatives of phenylpropane (mixtures of aldehydes, alcohols, phenols, methoxy derivatives and methylenedioxy compounds), for example, eugenol and cinnamaldehyde.5,6\n\nThe mechanisms of EO antimicrobial action are mediated by a series of biochemical reactions and depends on the type on their chemical constituents. Gram-positive bacteria are not considered as resistant as Gram-negative bacteria.6 This is attributed to differences in their cell wall structure, namely a thick peptidoglycan cell wall that allows penetration by phenolic compounds (for example, thymol, carvacrol, eugenol) present in EOs.7 The hydrophobic characteristic of EOs grant them a greater accessibility to the cell wall of Gram-positive bacteria, which is rich in peptidoglycans and unable to resist the presence of small antimicrobial molecules, leading to variations in the structure of the cell membrane. Consequently, this leads to cell lysis and leakage of intracellular compounds.8 These changes in the permeability of the cell wall and cytoplasmic membrane affect bacterial spread. Bacteria, in the presence of antimicrobial agents, alter the lipid profile of their membrane to incorporate exogenous fatty acids (from EOs), modifying the ratio of fatty acids, or the length of their carbon chains, and can increase the amount of saturated, or decrease the amount of unsaturated fatty acids. This reorganisation of fatty acids and membrane proteins allows for the survival of bacterial cells.6 However, the synergistic action of different EO constituents favours the death of the bacterial cell.9,10\n\nThe use of some EOs has been considered as an antimicrobial alternative, and has attracted considerable interest from the pharmaceutical industry, especially for its antifungal and antiviral activity.11â14 Phytochemicals present in EOs are also being evaluated as inhibitors of COVID-19.15 In the food industry, EOs are used for food preservation, due to their natural antimicrobial compounds against pathogenic bacteria, as well as aroma and flavour.16,17\n\nThe Bacillus species are included among Gram-positive bacteria. They are aerobic or facultative anaerobic bacteria that are widely distributed in nature.18 B. subtilis is considered as an aerobe bacterium, although it is able to sporulate under anaerobic conditions and starts to proliferate and grow in different environments including water, processed and untreated foods.19 Nowadays, the bakery industry is growing, giving rise to an increased demand for quality, new products developed, and for the extension of productsâ shelf life and safety. Previous studies have considered the use of EOs as antifungals to prolong bread shelf life.20 Research results from Pepe et al. (2003)21 have determined that after a 10-minute treatment at 96°, Bacillus spp. were able to survive the thermal processing of bakery products. Contamination of the dough by spores can occur via flour or the reuse of dried and ground bakery.22 The prevalence of spore-forming string-producing microorganisms in different types of flour and their potential for bread spoilage has been investigated; Bacillus spp. spores exhibit a high heat resistance and regularly survive the baking process inside the bread. Due to the action of proteolytic and amylolytic enzymes released by Bacillus, the texture of the bread is modified and becomes viscous.22 One of the species considered to be the most common causative agent of rope in bakery products is B. subtilis,23 however, as it takes a relatively long time for the stiffness to develop, the deterioration is often only detected once the consumer has purchased the product. There is increasing interest in developing new strategies to inactivate spores. EOs contained into microcapsules that gradually release volatile compounds into the packaging environment have been used in food preservation.24â26 Still, there is little information on the application of EO-containing microcapsules in the preservation of baked products.\n\nThe aim of this study was to evaluate the effect of 24 chemotyped essential oils on Bacillus subtilis growth and spore production, for potential use in the bakery industry, to answer a growing demand for high quality and extended shelf-life products, and the increasing trend of consumer demand for clean labels and minimum processing or synthetic preservatives. EOs were chosen based on their commercial availability, scent, and absence of toxicity in view of a possible bakery industry use. All of them are used traditionally as food additives. Of all EOs, Cymbopogon martinii var. motia was found to be an important source of natural geraniol. According to the literature review carried out by Lira et al. (2020),27 geraniol presents many pharmacological properties including antifungal and antibacterial actions. A secondary objective was to investigate the combined antibacterial activities of Cymbopogon martinii var. motia (palmarosa oil), Mentha x piperita (peppermint) and Eugenia caryophyllus (clove) essential oils against B. subtilis.\n\n\nMethods\n\nBacillus subtilis subsp. subtilis (Ehrenberg 1835) Cohn 1872, from the SpanishType Culture Collection (CECT), listed as CECT 4522, was used in this study. B. subtilis was maintained on a nutrient broth medium and solidified. The growth temperature was 30°C and the incubation time was 24 h. The nutrient broth medium contained 0.5% of beef extract (Laboratorios Condalab S.L., Madrid, Spain), 1% of peptone (Laboratorios Condalab S.L., Madrid, Spain) and 0.5% of NaCl (Merck Life Science S. L, Madrid, Spain); after mixing and dissolving them completely, the medium was adjusted to pH 7.2.\n\nChemotyped EOs were extracted from different plants by steam distillation by the supplier before purchase (PranarÃŽm, S.A., 7822, Ath, HAINAUT Belgium). The 24 EOs used for the study were: Citrus sinensis (sweet orange), Citrus reticulata (Mandarin orange), Elettaria cardamomum (cardamom), Laurus nobilis (laurel), Cymbopogon martinii var. motia (palmarosa), Zingiber officinale (ginger), Eugenia caryophyllus (clove), Cinnamomum camphora (camphor tree), Rosmarinus officinalis (rosemary), Melaleuca quinquenervia (niaouli), Chamaemelum nobile (Roman chamomile), Melaleuca alternifolia (tea tree), Thymus vulgaris CT LINALOL (thyme), Citrus paradisi (grapefruit), Citrus junos (yuzu), Origanum compactum (oregano), Mentha x piperita (peppermint), Myrtus communis (myrtle), Curcuma longa (curcuma), Cinnamomum cassia (Chinese cinnamon), Thymus satureioides (savoury thyme), Eucalyptus radiata (eucalyptus), Cinnamosma fragrans (saro) and Mentha arvensis (wild mint). Their chemical components are shown in Table 1.\n\nThe table shows the essential oils analyzed: common name, part of the plant and a percentage of some chemical components of selected essential oil after being subjected to steam distillation (data obtained from analysis sheet of PranarÃŽm; https://pranarom.us/products/essential-oils/).\n\nThe assessment of the antibacterial activities of EOs was performed using the disk diffusion method.28 Bacterial inoculum was measured using a spectrophotometer (Libra S12, Biochrom Ltd, Cambridge CB4 0FJ, England) set at 600 nm, and 0.20 mL at DO 0.45 were transferred to the plates.\n\nThe disk absorption capacity was 5 ÎŒL/disk. Three different concentrations of EO extracts were aseptically transferred to these disks to establish their antimicrobial activity. Sterile disks were impregnated with 5 ÎŒl of EO at different concentrations by serial dilution in vegetal oil (100%, 10%, 1%) (v/v) and each disk was placed on a nutrient broth agar plate smeared with B. subtilis. Every dish was sealed with laboratory film to avoid evaporation, then incubated aerobically in an upright position at 30 °C for 24 h to determine the antimicrobial effect. Antibacterial activity was determined by measuring the inhibition zone diameter (mm) against each EO. A sterile vegetal oil without EO was used as a negative control, and 5 ÎŒg of Ciprofloxacin (Thermo Fisher Scientific, Waltham, MA, USA) as positive control (dc).29 All experiments were done in triplicates.\n\nThe percentage of bacterial growth inhibition of B. subtilis was determined by considering the diameter of inhibition bacterial growth with EOs (dEO) referenced to the diameter inhibition bacterial growth with Ciprofloxacin antibiotic (dCIP), according to the following equation (Equation 1)\n\nOnce the results of the antimicrobial activity against B. subtilis, obtained by disk diffusion, were analysed for the 24 EOs tested, ten EOs were selected (C. cassia, C. sinensis, C. martinii var. motia, E. cardamomum, E. caryophyllus, M. x piperita, T. vulgaris CT LINALOL and T. satureioides). This selection also considered the principal chemical component in their composition. A B. subtilis inoculum (DO600 = 0.350) was incubated into the tubes containing different concentrations of these selected EOs (10 ÎŒL/mL, 25 ÎŒL/mL, 50 ÎŒL/mL, and 75 ÎŒL/mL). The tubes were incubated at 30 °C for 24 h and bacterial growth was measured using a spectrophotometer (Libra S12, Biochrom Ltd, Cambridge CB4 0FJ, England) at 600 nm.30 Vegetal oil was tested as negative control, at the same volume as the EOs.\n\nThe percentage of B. subtilis bacterial growth inhibition was determined by considering the optical density of bacterial growth without EOs (ODc) minus the optical density of bacterial growth with the presence of EOs (ODp), divided by the optical density of bacterial growth without EOs (ODc) (Equation 2).\n\nThe interaction studies (I) of the major components of EOs were performed using the agar disk diffusion method. The binary mix 1:1 (v/v) of EOs or their components, absorbed on sterile paper disks (5 ÎŒL per Whatman disk of 5 mm diameter), were placed on the surface of media that had previously been inoculated with 200 ÎŒL of B. subtilis (DO600 = 0.45). One filter paper disk was placed in each Petri dish, which was sealed with laboratory film to avoid evaporation, then incubated aerobically at 30°C for 24 h, followed by measurements of the diameter of the inhibition zone in cm.\n\nThe interaction was calculated as IA + IB, where IA and IB are the diameter of the inhibition zone (D) for EOs A and B, respectively. Thus, it was calculated as follows: IA = (DA combination / DA alone) and IB = (DB combination/DB alone). The results were interpreted as antagonism (I < 0.5) or indifference (0.5 †I †1). All experiments were done in triplicates.\n\nData were recorded using Microsoft Excel, which was also used for graphic data representation. Statistical analysis was performed using SPSS v27.0 software (SPSS Inc., Chicago, IL, USA). Normality of the dependent variables was checked using the Shapiro-Wilk test. If normality was observed, a one-way analysis of variance (ANOVA) was carried out. If normality was not observed, the Kruskal-Wallis test was used. The variability among the three measures was reported with the coefficient of variation. The reliability of the three measures (Underlying data) was reported with the intraclass correlation coefficient (2.1) looking for absolute agreement. A significance level of 0.05 was used. Values are reported as the mean ± standard deviation (SD).\n\n\nResults\n\nEOs are complex mixtures of volatile compounds; their main components are described in Table 1. The information was obtained from a data sheet provided by delegates from PranarÃŽm, S. A (Spain) and available on the PranarÃŽm website. As shown in Table 2, these volatile molecules include terpenes (hydrocarbon and oxygenated monoterpenes), terpenoid (oxygen atoms added to the hydrocarbon molecules), phenylpropene (phenyl group attached to an unsaturated aldehyde or ether), alcohol terpene (saturated secondary alcohol) and sesquiterpenes (hydrocarbon and oxygenated sesquiterpenes).\n\nThe essential oils have been grouped into five classes of considering the chemical structure of their major component.\n\n1 Values are mean diameter of inhibitory zone (cm) ±SD of three replicates. The diameter of paper disk (0.5 cm) is included.\n\nIn the terpenes analysed, limonene, a monocyclic monoterpene, was present in Citrus junos, C. paradisi, C. reticula and C. sinensis; γ-terpinene was present in C. junos and C. reticula, highlighting the presence of β-phellandrene in C. junos. Geraniol was the most abundant compound in the EO of C. matinii var. motia, along with a significant percentage of geranyl acetate, and the presence of a bicyclic sesquiterpene, β-caryophyllene, as well as linalool (terpene with an alcohol group). The ether monoterpene 1,8-cineole, was present at a high percentage in C. camphora, C. fragrans, E. radiata, L. nobilis, E. cardamomum, M. quinquenervia and M. communis. Terpinene-4-ol and Ê-terpinene, were identified in M. alternofolia, while Rosmarinus officinalis was characterized by α-pinene, followed by camphene, camphor and bornyl acetate. M. communis contained monoterpenes such as α-pinene, 1,8-cineole and limonene.\n\nAmong the main terpenoids identified in the EOs of O. compactum and T. satureioides were carvacrol and thymol; in contrast, borneol and β-caryophyllene were only present in T. satureioides. Thymus vulgaris contained linalool while and linalyl acetate. The main constituents of C. nobile were methylamine angelate, metalyl angelate and hexyl-isobutyrate.\n\nMajor components of C. longa belong to the chemical class sesquiterpenes which includes α-turmerone, β-turmerone, curlone and AR-turmerone. In addition, within this group, zingiberene is a monocyclic sesquiterpene and the predominant constituent of the oil of Z. officinale. Eugenol, hydroxyphenyl propene, was present in the essential oils of E. caryophyllus. Cinnamaldehyde is an aromatic aldehyde and main component of bark and leaf extract of C. fragrans. An additional terpene alcohol analysed belonged to M. x piperita and M. arvensis. The major chemical constituent of M. x piperita were menthol, menthone and 1,8 cineole, which are all cyclic monoterpenes. The main difference between these molecules is the presence of different functional groups: alcohol, ketone, and ether respectively. In addition, the difference with the EO of M. arvensis was the presence of isomenthone and limonene instead of 1,8-cineole.\n\nThe results of this study show that not all tested EOs had the same activity against the growth of B. subtilis. Using the disk diffusion method, the Gram-positive bacteria under study was inhibited by all the citrus oils tested (Table 2), which confirms previous works.31,32 When comparing these terpene EOsâ antimicrobial activity using the agar-disk diffusion assay, we observed that the effectiveness of limonene action increased when other terpenes were mixed, which is evidenced by contrasting the inhibitory results against the bacteria from the action of Citrus sinensis and C. junos. Although there is different concentration of limonene in both EOs (Table 1). These synergistic antimicrobial effects of the isolated compounds from C. limon against Gram-positive bacteria and B. subtilis also was observed by Nsangou et al. (2021).33\n\nC. martinii var. motia exhibited the second most potent antibacterial activity after O. compactum, among all tested EOs (Table 2). The antibacterial activity of O. compactum has been related to the presence of two aromatic terpenoids: carvacrol and thymol34 as opposed to geraniol, an acyclic monoterpene alcohol,27 the main ingredient of C. matinii var motia (Table 1).\n\nWe observed the antimicrobial activity of seven EOs (C. camphora, C. fragrans, E. radiata, L. nobilis, E. cardamomum, M. quinquenervia and M. communis) in which the terpene 1,8-cineole (Table 2) was present in relatively high concentrations. E. cardamomum showed the greatest inhibitory effect. A similar antimicrobial activity on the agar disks was observed for C. camphora, L. nobilis and M. quinquenervia. The difference in the antimicrobial activity between them may be due to the presence of some terpenes in their composition and their synergistic effects.36 For example, E. cardamomum, L. nobilis and C. camphora showed a greater antimicrobial activity, with the former containing α-Terpenyl acetate in greater proportions than L. nobilis, and C. camphora containing sabine (Table 1).\n\nThe remarkable difference between the chemical composition of M. alternofolia and M. quinquenervia, and their similar effect against the growth of B.subtilis, may be due to the presence of terpinene 4-ol37 in the former, and the presence of 1,8-cineole in the latter. The chemical components α-pinene and bicyclic terpenes could be found in the essential oil of Rosmarinus officinalis (Table 1), whose antimicrobial activity was greater than that of Myrtus communis. Rosemary oil also contained other major components, such as camphene, camphor and bornyle acetate37; their synergistic effect gives Rosemary oil a greater antimicrobial activity.38\n\nIn an agar-disk diffusion assay (Table 2), cinnamaldehyde from Cinnamomum cassia (Chinese cinnamon oil) showed a higher antimicrobial activity against B. subtilis than eugenol from Eugenia caryophyllus (clove oil). As presented in Table 2, the EO of Cinnamomum cassia showed an antibacterial activity comparable to that of Origanum compactum oil, preventing the growth of bacteria. According to the results reported in Table 2, it seems that the synergistic action of carvacrol and thymol from O. compactum is comparable to the activity of cinnamaldehyde.\n\nThe terpenoid group included T. vulgaris and T. satureioides oils. The former contained linalool (Table 1), which from the inhibition zone showed less inhibitory activity than T. satureioides (Table 2). The action of borneol present in T. satureioides led to a greater growth inhibition of the bacteria through the synergistic action of carvacrol and thymol (Table 2) present in O. compactum. These bioassays by disk diffusion showed a similar inhibition growth activity in O. compactum and Cinnamomum cassia EOs.\n\nMenthol, the dominant compound present in M. arvensis and M. x piperita oil (Table 1), showed a moderate antibacterial activity against B. subtilis growth (Table 2) using the disk diffusion method.\n\nMost of the undiluted, commercially available EOs used, gave rise to an inhibition zone against B. subtilis. Among the 24 oils tested, it was observed that their antibacterial effects were reduced at lower oil concentrations, suggesting that the inhibition halo was dependent on each essential oil.\n\nThe antimicrobial action of these EOs, as shown by agar-disk diffusion tests, was comparable to the activity of the antibacterial ciprofloxacin (Table 2), while the vegetal oil control did not affect the growth of bacteria. The assessed antibiotic ciprofloxacin was selected for its antibacterial effect, previously described by Citron and Appleman (2006)29 showing antimicrobial action against B. subtilis spores. To this end, EOs were arranged into five groups according to their main chemical component responsible for their inhibitory activity: terpen, terpenoid, alcohol terpenoids, sesquiterpenes and phenylpropanoids (Figure 1A). Figure 1B shows the activity of chemical phenolic groups with respect to the other chemical groups. These results showed the following order in antimicrobial efficiency in agar diffusion disk assays: phenylpropene > terpenoid > alcohol terpene > terpene > sesquiterpene, without reaching statistical significance. It was found that the oils containing alcohol, ketone, ester, oxide, and hydrocarbon as major constituents showed high antimicrobial activity, but even higher antimicrobial activity was found in the oils containing phenol or phenyl derivatives that contain aldehyde and methoxy groups exhibited the highest antibacterial activity. This activity was previously observed by other authors.6,39 Table 3 shows the reliability, referring to the consistency of the three diameter measures, relating to the zone of growth inhibition of B. subtilis (H), and the data when comparing the activity of the 24 EOs at three different concentrations, with the inhibitory action of Ciprofloxacin (HA) against the same bacterium. Reliability values closer to 1 represent a stronger reliability, however, it decreases as EOs are diluted.\n\nIn the left side, the five chemical components groups of the EOs under study. In the right side, the graphical corresponding to the medium values of agar diffusion disk activity against B. subtilis growth for each EOs group. (*) differences with respect to terpenes with p < 0.05; (#) differences with respect to sesquiterpenes and alcohol-terpenes with p < 0.05.\n\nValues of reliability from three replicates of measures of the diameter of inhibitory zone B. subtilis growth (H) and the activity respect to the action of antibiotic Ciprofloxacin (HA) of the 24 EOS at three different concentration.\n\nThe results obtained in this study from inhibitory diameters and concentrations of 24 EOs, revealed that ten of them showed higher inhibition against B. subtilis growth (Figure 2): Cinnamomum cassia (Chinese cinnamon), Citrus sinensis (sweet orange), Cymbopogon matinii var. motia (palmarosa), Elettaria cardamomum (cardamon), Eugenia caryophyllus (clove bud), Mentha arvensis (field mint), Mentha x piperita (pepper mint), Origanum compactum (oregano), Thymus vulgaris CT Linanol (thyme) and Thymus satureioides (thyme with savoury leaves). These EOs represent four of the five earlier described groups (Table 2); each of them containing different chemical components found to have high antimicrobiological action by Lira et al. (2020),27 Fisher and Phillips (2006),32 Laghmouchi et al. (2018),34 Mulyaningsih et al. (2010)35 and Bassolé and Juliani (2012).40 To analyse their antimicrobial activity in aqueous solutions, the Minimum Inhibitory Concentration (MIC) of EOs was determined as described in our Methods. Table 4 shows the inhibitory activity of ten EOs against the growth B. subtilis, at four different concentrations (v/v) in nutrient broth culture. The lowest MIC values were found for cardamon, sweet orange, field mint and palmarosa oils. The lowest antimicrobial activities were observed for thymus, Moroccan thyme, peppermint and clove bud oil. The activity of Chinese cinnamon and compact oregano oil in aqueous solutions were lower than that observed using the diffusion method, where the EO is laid on a paper disk. After a 24 h-incubation period of B. subtilis in the presence of Origanum compactum oil, a colour change was observed in the culture broth medium; this may be due to the presence of carvacrol, which increases permeability of the bacterial membranes and releases H+ into the culture medium.6,41\n\nLikewise, the major and biologically bioactive constituents present in the six essential oils are shown.\n\n1 Values are results of two experiments with two replicates.\n\nAfter comparing the antibacterial activity of these ten EOs against B. subtilis using disk diffusion and broth dilution methods, and considering their antimicrobial components, we focused on Cymbopogon matinii var. motia, the main component of which is geraniol. Palmarosa oil, with specific rose fragrance,42 appears to be a good candidate to be used as an antibacterial agent against Bacillus subtilis in the bakery industry. Table 5 shows the results of the synergestic antibacterial activity of Cymbopogon matinii var. motia combined with each of four EOs: Eugenia caryophyllus, Mentha arvensis, Mentha x piperita and Thymus vulgaris CT Linalol. For this study, we carried out disk diffusions using a 1:1 (v/v) combination for C. martini var. mortia and each EO selected. The results showed that three combinations of EOs with different bioactive components displayed an insignificant antibacterial activity against these Gram-positive bacteria: geraniol/menthol, geraniol/menthol-menthone, geraniol/linalool. Only the geraniol/eugenol combination showed an antagonistic effect.\n\nFractional inhibitory concentration and interaction among essential oil major components\n\n1 I. indifference; A. antagonist.\n\n\nDiscussion\n\nEOs are composed of a mixture of complex, low-molecular-weight organic compounds such as terpenoids, phenolic acids, flavonoids, and phenylpropanoids.39 They represent a natural source of bioactive compounds. Their constituents play a key role in antimicrobial activity, through properties which are toxic for bacteria and other microorganisms. For example, the phenolic content causes disruption of plasma membrane structure and alters the membrane permeability43; terpenes and terpenoids alter the permeability of the plasma membrane when interacting with their fatty acids, allowing for the release of cytoplasmic constituents.7,44 All the EOs tested showed antibacterial properties against the Gram-positive bacterium B. subtilis (Table 2), some of them having a weaker antibacterial effect than others. The bioactive components 1,8-cineole (present in: ravintsara, saro or mandravasarota, Eucalyptus officinalis, laurel noble and niaouli oils), α-pinene (present in: cineole blueberry and verbenone rosemary oils), α-zingiberene (present in ginger oil), limonene (present in: yuzu junos, grapefruit, mandarin and sweet orange oil), sabinene (present in: ravintsara and laurel noble oil) are known for their weak antibacterial activity, compared to alcoholic and phenolic monoterpenes such as carvacrol and thymol (present in Origan compact oil).34,45 The monocyclic monoterpenoid compound terpinen-4-ol has been shown to inhibit B. cereus biofilm formation46 and Staphylococcus aureus36 but showed a weak effect against B. subtilis. The antibacterial activity of the Cymbopogon martinii var. motia EO (palmarosa oil) could be related to its high levels of geraniol, an acyclic monoterpene alcohol. The antimicrobial activities of EOs appear to be related to their chemical composition, and our results corroborate previous studies showing their antimicrobial activity against the Bacillus genus from the work by Syed et al.47\n\nCinnamomum cassia and Origanum compactum oils showed more activity at higher concentration (no dilution, 100%) in agar-disk diffusion assays; according to the results (Table 2) the former EOâs antimicrobial action may be associated with the presence of high contents of E-cinnamaldehyde that can be compared with the antibacterial activity of the carvacrol and thymol present in O. compactum oil. This was previously described by Helander et al. (1998) in relation with the inhibitory activity against Escherichia coli.48 Regarding the antimicrobial activity shown by sesquiterpenes, which are the main compounds of Curcuma longa, their aromatic group leads to hydrophobicity. Their poor water solubility may explain the lower antibacterial activity against B. subtilis as described by TÞnnesen et al. (2002).49 The antimicrobial activity of oil constituents from C. nobile (Table 2) was previously described by Piccaglia et al. (1993)50 and is known for its therapeutic uses, especially through its binding to different cell receptors involved in several biochemical pathways, related to inflammation and several metabolic disorder.51 Our results showed around 26% inhibition of the growth of B. subtilis, when compared with the antibacterial activity of antibiotic ciprofloxacin, which acts at the level of the bacterium DNA.52 Zingiber officinale essential oil has been used as a natural food additive and preservative, and previous studies have shown that the strongest antibacterial effect of this EO was observed against B. subtilis53; this corroborates our results. The twenty-four EOs tested with different major chemical components (Table 1) had different degrees of growth inhibition against B. subtilis, and their antibacterial activities were reduced at lower oil concentrations; according to the results (Table 2) the inhibition halo was dependent on each essential oil.\n\nAfter analysing EO antimicrobial effect using agar-disk diffusion assays, we focused on 10 EOs (Figure 2) to evaluate their antibacterial activity in aqueous solution, measured as MIC (Table 4). These EOs showed antimicrobial activity against B. subtilis, with a wide range of inhibition values; the lowest MIC values were found for cardamon, sweet orange, field mint and palmarosa oil. Low antimicrobial activity was observed for cimbru, thyme with savoury leaves, peppermint and clove bud oils. The hydrophobic characteristic of some compounds from the EOs (E-cinnamaldehyde, limonene, carvacrol, linalool, thymol, borneol) can explain the difference in antibacterial activity between broth medium culture and disk-diffusion method, for some EOs such as Chinese cinnamon and oregano compact oils.\n\nThis study has conducted an analysis about the antibacterial activity of the EOs against B. subtilis (Tables 2 and 3). Taking into consideration the previous results from other authors in this matter,54 we set a goal to perform a specific study for C. martinii var. motia, EO with a high geraniol content (Table 1) to which different biological activities have been attributed (antimicrobiological, antioxidant and anti-inflammatory) by MÄ
czka et al.,55 highlighting the fact that EOs contains major and minor chemical components, and their combination can contribute to their antimicrobial properties. However, due to the impact of the taste and scent of some EOs, their application as food preservatives is not fully extended; therefore, the combination of different EOs is an alternative to improve these effects as well as to reduce their organoleptic impact in food. It was suggested that some mixtures of these EOs could be determining synergistic, antagonistic, or absence of interactions between them against bacterial growth.56,57 Studies on the antimicrobial activity of EO associations were developed using binary combination at the same proportion (v/v), using the disk diffusion method, which consisted of four binary combinations of C. martinii var. motia with E. caryophyllus, T. vulgaris CT Linalol, M. arvensis and Mentha x piperita EOs. The results of the combined effect of blending of monoterpene alcohols (geraniol, linalool), cyclic terpene alcohol (menthol), phenylpropanoids (eugenol), is showed in Table 5. The geraniol/eugenol combination showed antagonistic effects on the growth of B. subtilis, while geraniol/menthol and geraniol/linalool showed no effect. Eugenia caryophyllus did not show an antimicrobial effect when it was tested independently (2.5 ÎŒL). However, the study carried out by Galluci et al. (2009),57 showed that the geraniol/menthol combination exhibited a high antimicrobial activity against B. cereus, and the synergistic antimicrobial activity of geraniol/eugenol was partially efficient against the bacteria.\n\nPathogen control in the food industry is the key to ensure food safety,58 including bakery products, which play important roles in human health and diet.59 Previous authors have also shown the antibacterial activity of monoterpenes were present in EOs, and their potential use for the food industry.20,25,60 Geraniol is non-polar, making it more able to permeate the lipid structure of microorganic cell membranes, causing K+ leakage from Saccharomyces cerevisiae.61 Considering that geraniol is the principal components of C. martinii var. motia (> 80%), together with geranyl acetate, linalool and b-caryophyllene, it appears to be a good alternative as an additive.\n\nFresh dough is a type of product readily susceptible to microbial deterioration; however, many of the chemicals licensed for use as food preservatives are being questioned regarding their effects on human health. The bakery industry tries to control microbiological spoilage by following several strategies including reformulation of the product and incorporating some preservatives; this is not an easy task, as microorganisms are found in the air or in the water. There are few research reports on EO applications in bread or other bakery products, or the impact their addition can have on dough and bread production, on physico-chemical, microbiological, and taste aspects. The spoilage of bakery products may occur through microbiological contamination.\n\nRope formation is a serious, but underreported food security problem in the bakery industry. Although this problem has been recognized for many years, effective means of prevention have not yet been determined. B. subtilis is one the bacteria responsible for rope spoilage in bread preservation processes.62,63 Recent studies have revealed the antibacterial activity of different EOs applied in bakery products including thyme, cinnamon, oregano, and lemongrass, that can inhibit the growth of harmful microorganisms, resulting in a product with extended shelf-life and enhanced safety.63 Palmarosa oil is a good alternative as an additive in the bakery industry since in addition to its antimicrobial activity, its volatility has been previously shown to cause the reduction of approximately 60% of the geraniol component over 24 hours.61 A controlled liberation of the EO and the high evaporation rate of geraniol may avoid lethal damage of baking yeast during the bread-making process.\n\nNanotechnologies offer very interesting prospects for food industry, the 'novel food' includes innovative food, as well as food produced using new technologies and production processes. References to nanotechnology and nanomaterials in European regulations are scarce, especially to the use of essential oils as preservatives for bakery products.64 The use of palmarosa oil could be considered to avoid the presence of ârope in breadâ, considering their activity against B. subtilis and its pleasant fragrance, which can improve the flavor of the product. At the same time, the palmarosa essential oil plays an important role as an antioxidant in food, thus preventing potential health risks associated with microbial contamination. It is highly reactive against free radicals from reactive oxygen species generated by a wide variety of sources in biological systems.65 Microencapsulation using EOs presents the advantage that it maintains the effectiveness of antimicrobial activity through the gradual release of the active components of EO, from the capsules to the bakery product. We must not ignore other possible uses of palmarosa oil, and its high proportions of geraniol, to increase the safety of bakery products, such as essential oil-loaded films, analysed by Agarwal et al. (2020),66 which can be used in active bakery ingredients such as those used in sourdough.\n\n\nConclusions\n\nThe antimicrobial activity of EOs depends on their composition in volatile compounds, such as terpenes, terpenoids, phenol-derived aromatic components and aliphatic components. They represent a natural source of bioactive compounds. A total of 24 EOs, with different compositions, have been analysed.\n\nThe EOs tested showed different antibacterial effects against the growth of Bacillus subtilis. These antibacterial activities were reduced at lower oil concentrations; thus, the result from the disk diffusion tests suggest that the antibacterial activity of each EO is dose-dependent. These results show the following antimicrobial activity in order of efficiency in agar diffusion disk assays: phenylpropene > terpenoid > alcohol terpene > terpene > sesquiterpene. It was found that the oils containing alcohol, ketone, ester groups and hydrocarbon as major constituents exhibited a greater antimicrobial activity, whereas the oils containing aldehyde or methoxy groups covalently linked to aromatic organic compounds such as phenyl and phenol groups, exhibited dominant diffusion activity.\n\nThe results obtained in this study confirm that Cymbopogon martinii var. motia, which contains geraniol, a compound with antioxidant effects, may be used to prevent the growth of B. subtilis, responsible for ârope formationâ in the bakery industry. The formulation of palmarosa/clove bud EOs tested in this study have an antagonistic effect against the growth of B. subtilis.\n\nThe possible use of palmarosa oil, as a potential natural solution to increase the shelf life and safety of bakery products, brings new technological solutions. With the development of techniques such as nanoencapsulation for bakery doughs, active packaging of baked products or new surface disinfectants, the Cymbopogon martinii var. motia essential oil can be an alternative in the bakery industry due to his high evaporation rate and organoleptic effect.\n\n\nData availability\n\nFigshare: Antibacterial activity of EO against Bacillus subtilis, https://doi.org/10.6084/m9.figshare.15129057.67\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC BY 4.0 Public domain dedication).",
"appendix": "Acknowledgments\n\nThe authors would like to thank Alfredo Quevedo Fernandez and Francesca Falzone from PranarÃŽm (Spain) for providing information and the analysis sheet of the essential oils; we also acknowledge the statistical support given by Azael J. Herrero.\n\nDisclaimer: a part of the present report has been presented in a preliminary form at Proceedings 2020, 66, 1; doi:10.3390/proceedings2020066001.\n\n\nReferences\n\nBurt S: Essential oils: Their antibacterial properties and potential applications in foodsâA review. Int. J. Food Microbiol. 2004; 94: 223â253. PubMed Abstract | Publisher Full Text\n\nDudareva N, Negre F, Nagegowda D, et al.: Plant Volatiles: Recent Advances and Future Perspectives. Crit. Rev. Plant Sci. 2006; 25: 417â440. Publisher Full Text\n\nArumugam G, Swamy MK, Sinniah UR: Plectranthus amboinicus (Lour.) Spreng: Botanical, Phytochemical, Pharmacological and Nutritional Significance. Molecules. 2016; 21: 369. 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}
|
[
{
"id": "156436",
"date": "10 Jan 2023",
"name": "Daniel Rico",
"expertise": [
"Reviewer Expertise Food processing (novel technologies). Postharvest. Bioactivity (in vitro) determination of food matrices and ingredients."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work titled âEssential oil of Cymbopogon martini, source of geraniol, as a potential antibacterial agent against Bacillus subtilis, a pathogen of the bakery industryâ, and submitted for publication to the F1000Research journal, is focused on screening the antibacterial effect against B. subtilis of a number of chemotyped essential oils.\nThe experimental design is appropriate to the objective intended, and the experimental methodology sufficiently described in detail. Antimicrobial activity of a significant number of essential oils against B. subtilis is presented, and discussed based on the main components present in these EOs. Interesting potential of the Cymbopogon martinii var. motia essential oil is concluded by the authors.\nI find the work suitable for indexing after minor revision. Please check comments below.\nSection Results/Chemical composition of EOs (PranarÃŽm [S.A]). Since the results showed in this subsection are a compilation of the information provided by the essential oil manufacturer, I would recommend the authors to reduce the extension of this part, and keep the minimal extension to fulfil the main purpose of classification of the different EOs used based on their main compound. I would also recommend to avoid the use of terms such as âanalysedâ or âidentifiedâ, which could create some confusion on the origin of the results of this subsection.\nSubsection âDetermination of minimum inhibitory concentration of essential oils against B. subtilisâ (in Methods, Page 4). Regarding EOs selection of 10 for the second part of the experimental work, the authors have stated that âThis selection also considered the principal chemical component in their compositionâ. Could the authors please be more specific and clarify the criteria for this selection of 10 EOs? Many thanks.\nIn table 2, the description given of the second calculated parameter, â% B. subtilis growth inhibition against Ciprofloxacinâ, seems a bit confusing. Please provide a clearer definition, as it appears that a 0% should correspond to no inhibition at all, as it says growth inhibition, when from the equation and diameters of inhibition areas, it appears 0% corresponds to 100% inhibition (similar to ciprofloxacin).\nPlease specify in material and methods, section âCombined antibacterial effect of EOs using disk diffusion testâ, how values over 1 are classified, as some over 1 are shown in Table 4, and classified as âindifferenceâ\nSection âConclusionâ. It would be interesting to add some comments in regard the concentration of the EO that should be necessary for the applications (bakery) suggested. Also, a comment on one of the sentences in this same section. Please revise the sentence finishing ââŠgroups, exhibited dominant diffusion activityâ, and check if it conveys the intended meaning by the authors. Maybe the word âdominantâ should be checked. Thank you.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9370",
"date": "24 Mar 2023",
"name": "Maria Angeles Rojo",
"role": "Author Response",
"response": "Thanks for your advice and corrections. Below we provide responses to your observations. 1. âSection Results/Chemical composition of EOs (PranarÃŽm [S.A]). Since the results showed in this subsection are a compilation of the information provided by the essential oil manufacturer, I would recommend the authors to reduce the extension of this part, and keep the minimal extension to fulfil the main purpose of classification of the different EOs used based on their main compound. I would also recommend to avoid the use of terms such as âanalysedâ or âidentifiedâ, which could create some confusion on the origin of the results of this subsectionâ. The information in the section of results has been reduced. The terms \"analysed\" or \"identified\" have been replaced by \"assayed\" and \"evaluated\" respectively. 2. Subsection âDetermination of minimum inhibitory concentration of essential oils against B. subtilisâ (in Methods, Page 4). Regarding EOs selection of 10 for the second part of the experimental work, the authors have stated that â This selection also considered the principal chemical component in their compositionâ. Could the authors please be more specific and clarify the criteria for this selection of 10 EOs? âIn the section we have expanded the information which, in addition to taking into account our results, considers previously published results 3. âIn table 2, the description given of the second calculated parameter, â% B. subtilis growth inhibition against Ciprofloxacinâ, seems a bit confusing. Please provide a clearer definition, as it appears that a 0% should correspond to no inhibition at all, as it says growth inhibition, when from the equation and diameters of inhibition areas, it appears 0% corresponds to 100% inhibition (similar to ciprofloxacin).â The appreciation has been considered and we have changed \"% B. subtilis growth inhibition against Ciprofloxacin\" to \" Inhibitory activity of EOs against Ciprofloxacin (%)\". In addition, a footnote has been added referring to equation 1 in the methods section. 4. âPlease specify in material and methods, section âCombined antibacterial effect of EOs using disk diffusion testâ, how values over 1 are classified, as some over 1 are shown in Table 4, and classified as âindifferenceââ In the methodology section, it has been described in the wrong way. And therefore, the calculations reflected in Table 4 are erroneous. They have been corrected Actually, the combination effect is calculated: I=D/(DA+DB) Thank you, It has been corrected 5. âSection âConclusionâ. It would be interesting to add some comments in regard the concentration of the EO that should be necessary for the applications (bakery) suggested.â Thank you for your suggestion. In our study we only focused on the possibility of using EO for bakery, analysing its effectiveness on the growth of B. subtilis. But the proposal is interesting to extend the study, our group is in fact extending the analysis to bakery products. 6. âAlso, a comment on one of the sentences in this same section. Please revise the sentence finishing ââŠgroups, exhibited dominant diffusion activityâ, and check if it conveys the intended meaning by the authors. Maybe the word âdominantâ should be checked. Thank you.â Thank you for your correction, it has been added âactivity in disk diffusion methodâ"
}
]
},
{
"id": "160708",
"date": "16 Feb 2023",
"name": "Abubakar Sunusi Adam",
"expertise": [
"Reviewer Expertise Antimicrobial resistance"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDetails of some methods have not been fully addressed. For example, the author stated that they used nutrient broth agar plate for antibacterial activity of the essential oil which is not appropriate. As such, the author should know that the standard medium to be used for any sensitivity testing is Mueller Hinton agar. However, broth and agar are two different entity, both cannot be use concurrently.\n\nFurthermore, preparation of essential oil concentrations was not clearly explained.\n\nUnder discussion, the author should compare the current findings with related reports from other researchers. This is because, different bacteria have different response against antimicrobial agents, in which essential oils are inclusive.\nOtherwise, the authors did a good job.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9369",
"date": "24 Mar 2023",
"name": "Maria Angeles Rojo",
"role": "Author Response",
"response": "Thanks for your advice and corrections. Below we provide responses to your observations. 1. âFor example, the author stated that they used nutrient broth agar plate for antibacterial activity of the essential oil which is not appropriate. As such, the author should know that the standard medium to be used for any sensitivity testing is Mueller Hinton agarâ The remark proposed is right. In the present study âMueller Hinton agarâ was considered to be a non-selective medium; the purpose of our study was to analyze EOs activity under the same conditions as those where the bacteria were laboratory-grown and preserved. Commercial EOs were not to be filtered prior to their use on the disk diffusion microbioassay in order to keep their small-sized volatile components. Yet, it would be of the utmost interest to conduct the same trials concerning the biological activity in the standard medium to compare the outcomes regarding antimicrobial activity. 2. âHowever, broth and agar are two different entityâ It has been corrected in the text. 3. âpreparation of essential oil concentrations was not clearly explainedâ Thank you for your comment, it has been modified in the text. Furthermore, it can also be stated that we used the microdilution method, where essential oils were 10 or 100 fold diluted. The % unit  was used to indicate the volume of EO added. 4. âUnder discussion, the author should compare the current findings with related reports from other researchers. This is because, different bacteria have different response against antimicrobial agents, in which essential oils are inclusiveâ In the bibliography we located studies of the same bacterium under study where the authors searched for antagonistic microorganisms against B. subtillis [Pepe O, Blaiotta G, Moschetti G, Greco T, Villani F. Rope-producing strains of Bacillus spp. from wheat bread and strategy for their control by lactic acid bacteria. Appl Environ Microbiol. 2003 Apr;69(4):2321-9]. In view to the study of ropiness in bread, B. subtillis is the bacteria that, according to our preliminary bibliographic search, was the least used in the analysis against the action of EOs. Concerning the characteristics of C. martinii var. motia, no similar studies were found. Just to mention an example, a bacteria analyzed by different authors is S. aureus due to its production of different toxins and its implication in human health."
}
]
}
] | 1
|
https://f1000research.com/articles/10-1027
|
https://f1000research.com/articles/12-331/v1
|
24 Mar 23
|
{
"type": "Research Article",
"title": "Neutrophil/lymphocyte ratio and overall survival in patients with breast cancer: a cohort study in a Latin-American hospital",
"authors": [
"Nataly Briyit Huamán Córdova",
"Martha Sofia Cervera-Ocaña",
"Dante M. Quiñones-Laveriano",
"Jhony A. De La Cruz-Vargas",
"Nataly Briyit Huamán Córdova",
"Martha Sofia Cervera-Ocaña",
"Jhony A. De La Cruz-Vargas"
],
"abstract": "Background: Breast cancer is a disease of high mortality globally. Inflammatory markers have been proposed as prognostic indicators. Therefore, we seek to find an association between the neutrophil-lymphocyte ratio (NLR) and overall survival (OS) of patients with breast cancer in a Latin-American hospital. Methods: An observational, analytical, longitudinal survival study was conducted in 241 female patients with breast cancer, between 2012 and 2014. The dependent variable was OS, the independent variable was the NLR, and the intervening variables were age, clinical stage, and molecular subtype. The KaplanâMeier method was applied to generate OS functions, and the Cox regression to find crude and adjusted hazard ratio (HR). Results: The mean age was 56.1 years and59.8% of patients had an NLRâ¥3.According to the KaplanâMeier estimator, NLRâ¥3 (p<0.001), age>55 years (p=0.039), and clinical stage (p<0.001), were associated with a lower OS. In the multivariate analysis, the factors significantly associated to a lower OS were NLRâ¥3 (HRa: 2.00; CI 95%: 1.36â2.95), age>55 years (HRa: 1.64; CI 95%: 1.10â2.44), and clinical stage IV (HRa: 68; CI 95%: 2.28â20.20).\n\nConclusions: The inflammatory marker NLRâ¥3 was independently associated with a lower OS. Additionally, advanced stage and older age were associated with a lower OS.",
"keywords": [
"Breast Neoplasm",
"Survival",
"Inflammatory markers",
"prognostic factors"
],
"content": "Introduction\n\nBreast cancer represents 16% of all female cancers, causing 2.3 million cases and 685,000 deaths worldwide in 2020.1,2 In Latin America, this pathology is the most important cause of death by neoplasms among women, with nearly 92,000 deaths,3 while in Peru it represents the most common primary neoplasm among women.4 Therefore, it is fundamental to determine the factors associated to overall survival (OS), such as age, comorbidities, metastasis, and laboratory analytical determinations such as neutrophil-lymphocyte ratio (NLR).5,6\n\nWhile research exists on an international level that associates an elevated NLR with lower OS in patients with breast cancer, these were developed in populations with early-stage diagnosis, as opposed to countries with middle and low incomes, where a high proportion are diagnosed in advanced stages. Furthermore, differences exist in breast cancer phenotype, prevalence of oncological infections,7 and the presence of social risk factors such as marked poverty and reduced access to healthcare services between Latin-American countries8 and others.9 This highlights the need for studying the OS associated with breast cancer and its prognostic indices in this context.\n\nSince in developing countries, the rate of clinical oncologists per million inhabitants is below the recommended value,7 simple, accessible, true, and low-cost methods, such as NLR, are required to potentially improve OS in these patients. Some research in Latin America has studied the NLR as an adverse prognostic factor in patients with different types of cancer, with few related to breast cancer.10,11 However, these studies were conducted only in patients with triple-negative breast cancer, which, although more aggressive, is not the most frequent of those presented in the region; therefore, it is necessary to perform studies including other cancer subtypes such as Luminal A, Luminal B or HER 2. In this sense, the general objetive of this study is to determine the association between NLR and OS in patients with breast cancer in the gynecology department of Hospital Nacional Hipólito Unanue (HNHU), Lima, Peru between 2012 and 2014. The specific objectives were to evaluate the association between OS and stage, molecular subtype and age. We hope to find an association between the NLR and the OS, so that the higher the NLR, the lower the OS.\n\n\nMethods\n\nAn observational, longitudinal, analytical survival study was carried out in female patients diagnosed with breast cancer, seen in HNHU, between 2012 and 2014. The research protocol was carried out and uploaded to protocols.io platform, explaining step by step how the study was carried out.12 The exposed group consisted of those patients with high NLR and their OS was compared with that of the group of patients with normal NLR. Both cohorts were women who were diagnosed at HNHU between 1st January 2012 and 31st December 2014. Follow-up began at the time of breast cancer diagnosis and exposure was measured on the blood cell count that was measured at the same time as the diagnosis. The follow-up period was up to eight years. The limitations were considered to be those patients who stopped attending their follow-ups without dying or without giving notice of death in the medical record. HNHU is the only III-I establishment in East Lima, Peru, which offers specialized and comprehensive care to cancer patients, and is characterized by having highly qualified staff, innovative health technology, and high-quality standards. The oncology department has a chemotherapy room equipped with nine service areas, one procedure room, and another of oncological combinations; these services are offered to the entire population, mainly to the patients most in need from Seguro Integral de Salud (SIS).13 Data was collected from medical records during the months of January and February 2020.\n\nWe did not have a sample since we worked with the entire population, with a total of 241 female patients. We found a statistical power of 98% for this number of participants to find an expected OS difference, as reported by a similar study,14 of 86.2% for the exposed group and 97.9% for the unexposed group. This gave us a low probability of making a type 2 error when testing the hypothesis of our main objective, which was to compare the OS of those exposed and not exposed participants. To be included in the study, patients had to be over 18 years of age, with complete and follow-up clinical information from the moment of diagnosis until the last consultation or death. Patients with immune-suppressing diseases or a HIV/AIDS infection were excluded. Follow-up was performed retrospectively from medical assessments recorded in the medical history.\n\nThe outcome variable of this study was the OS of the patients diagnosed with breast cancer, time was measured in months, from the time of diagnosis until death or date of last follow-up. A participant was considered dead if it was labeled as such in the medical record and corroborated with the death certificate. Additionally, the exposure variable was NLR, which was measured from the first blood count obtained at the time of diagnosis that appears in the medical record. This rate is defined as the division of absolute neutrophil count over absolute lymphocyte count. The rate was categorized as low if NLR<3 and high if NLRâ¥3, according to EnrÃquez et al.,10 since said cut-off point predicted OS and complete pathological response in this study, in a very similar context. These measurements were made in the same way in the exposed and unexposed group.\n\nAs confounders variables, we considered age, categorized in age groups for descriptive analysis and dichotomized (>55 years), for survival analysis. Likewise, we took into account the clinical stage of breast cancer from the imaging analysis registered in the medical record at the time of diagnosis. The classifications were stage I: small and invasive tumor, with a capacity to spread to lymph nodes; stage II: cancer spread to lymph nodes without evidence of tumor in breasts; stage III: cancer spread to 4-9 axillary or internal mammary lymph nodes, a tumor greater than 50 mm may be found; stage IV: presence of metastasis.15 We also considered the molecular subtype classified as Luminal A, Luminal B, HER 2, and triple-negative.16 These variables were considered in the multivariate model only if they were associated with the bivariate analysis outcome.\n\nTo reduce the selection bias, participants were selected from the same hospital, which contributes to the groups having fairly similar clinical and social characteristics. In addition, the clinical history was reviewed exhaustively to avoid information bias. Finally, an analysis adjusted for confounders was considered to avoid erroneous conclusions.\n\nThe data collection technique and follow-up were carried out from the documentation, through a review of medical records of each patient and filled out in a data collection sheet. This research instrument, designed specifically for this study (Extended data33), was filled out with the patientâs information, such as age, date of breast cancer diagnosis, molecular subtype, clinical stage, absolute neutrophil count, absolute lymphocyte count, NLR, date of last follow-up, and, if applicable, the date of death and cause of death. Once data was collected, the clinical records were entered into a data matrix in the Microsoft Excel (RRID: SCR_016137), which was used as a database. Later, this data was used to obtain calculations and graphs through the Stata (RRID: SCR_012763) 15.0 statistical program.\n\nFirst, we found the general characteristics of the entire population using frequencies and percentages for the qualitative variables, and central tendency and dispersion measures for quantitative variables, prior evaluation of its normalcy using the Shapiro-Wilk test.\n\nLater the KaplanâMeier method was applied to generate survival curves, which were compared using the log-rank test. Likewise, Cox regression was used to find crude and adjusted Hazard Ratios, with its respective 95% confidence intervals. The multivariate model included, as confounding variables, those variables that had statistical significance in the bivariate model, since they could influence the outcome. A p-value<0.05 was considered statistically significant.\n\nThere was only one observed missing observation in the molecular subtype variable and no extra modifications were made before the analysis. This missing number was not relevant to the conclusions of the study as this variable was not included in the multivariate model. Subjects who did not complete all follow-up were also included in the analysis. No sensitivity analyses were performed.\n\nThis study was approved by the research ethical committee of Hospital Nacional Hipólito Unanue (file N° 39993; 12th December 2019. All the patientsâ data remained in absolute confidentiality by encrypting their personal identification. Patients, or their relatives, signed a written informed consent at the time of admission to the hospital, in favor of performing medical procedures and using their data for teaching and research purposes.\n\n\nResults\n\nOf the 324 initial medical records, we excluded 35 not found and 48 incomplete medical records, leaving a total of 241 medical records for analysis (Figure 1). A follow-up of patients was performed in a maximum period of 8 years, with a mean follow-up of 3.5±1.8 years total, 2.7±1.8 years in the exposed group, and 3.9±1.7 years in the non-exposed group. Seventy-five patients (31.1%) were diagnosed in 2012, 84 (34.9%) in 2013, and 82 (34%) in 2014, with 130 patients dying during the study (Table 1). The age range was 27 to 85 years old (mean: 56.1 years±11.6), the most frequent molecular subtype of breast cancer was HER2 (39.2%), while the most frequent clinical stage was II (42.7%). Furthermore, 144 (59.8%) patients had NLRâ¥3. The other general population characteristics are presented in Table 2.\n\nWhen evaluating OS according to the year of follow-up, we observed that patients with NLRâ¥3 had a lower OS (Figure 2A), compared to those who had an NLR<3. This association was statistically significant (p<0.001). When evaluating OS by molecular subtype, no statistically significant differences are observed (p=0.528) (Figure 2B). While in the clinical stage (Figure 2C), OS decreases in stage IV (p<0.001). Furthermore, we observed that the age group >55 years (Figure 2D) presented a lower OS within the follow-up years (p=0.017), compared to patients up to 55 years of age.\n\n(A) According to NLR. (B) According to molecular subtype. (C) According to stage. (D) According to age.\n\nIn the multivariate analysis, the NLRâ¥3 (p<0.001) was a risk factor for mortality, adjusted for confounder variables of age and clinical stage. Additionally, age (p=0.016) and clinical stage IV (p=0.001) were also risk factors for mortality (Table 3).\n\n\nDiscussion\n\nIn our study, we found that NLRâ¥3 (p<0.001), age>55 years (p=0.039), and clinical stage (p<0.001), were associated with a lower OS. In the multivariate analysis, the factors significantly associated to a lower OS were NLRâ¥3, age>55 years, and clinical stage IV. Although there have been international studies, we must consider that in all of them the population characteristics, such as risk factors, prevalence of infections, genetic susceptibility, and existing polymorphisms, are different from the Latin American context,9,17,18 which makes it difficult to generalize its results in this context. This study is especially important given that a broad follow-up of 8 years for the NLR evaluation is a prognostic factor of OS in patients in a Latin American hospital. In this regard, this type of cancer is considered a health problem of high priority,19 and the lack of resources requires cost-effective tools of easy access to orient the prognosis and need for treatment by a specialist.\n\nRegarding OS, we found that an NLR>3 was related to a lower OS. Similar results were found by a Peruvian study, only in breast cancer with a triple-negative molecular subtype, while in our study we evidenced the use of this ratio in other molecular subtypes. Similar findings have been found in studies carried out in other regions of the world.20 This is probably because a greater NLR reflects a greater number of neutrophils, whose high infiltration has been associated with the aggressiveness of the disease and resistance to therapy, in addition to promoting pro-cancerous factors and suppressing the cytotoxic activity of lymphocytes.21â23 On the other hand, a decrease in lymphocyte count may decrease the effectiveness of the anti-tumoral immune response, which is why the combination of these conditions reflected in the NLR seems to be an excellent prognostic tool. Furthermore, it can be obtained through an accessible exam such as blood count, which is why its incorporation into the established prediction of risk justifies a larger study.24\n\nWe observed that patients with NLRâ¥3 had a lower OS, however, as of the fifth follow-up year the survival curves in both NLR categories tended to approach each other. This is similar to other inflammatory indices such as the combination of the NLR with the lymphocyte platelet index (NLR/LPR) studied by Hirahara et al.25 They also observed that survival was greater as this rate decreased (p<0.0001); however, from approximately the fifth follow-up year, we observed that survival functions were similar. This indicates that NLR or other hematological rates are likely not useful in predicting OS after a certain number of years. Likewise, in a Peruvian study,11 the survival functions with an NLR<2.5 and NLRâ¥2 were different during a 2-year follow-up in patients with triple negative breast cancer. It is probable that if it they had a longer follow-up, the behavior of the curves would have been similar to our study. Breast cancer mortality after 5 years could be high due to diverse factors foreign to what NLR might reflect, such as tumoral growth, comorbidities, and age-related functional impairment.\n\nWe found that over half of patients had an NLRâ¥3, similar to that found in other studies.26 On the other hand, the stages that were most frequently found during diagnosis were stages II and III, unlike the United States, where the most frequent stages were I and II.27 This could reflect a late diagnosis, typical of developing countries with deficits in their health system.7 Conversely, the average age at the time of diagnosis is comparable to that of other international reports.28 The most frequent molecular subtype was HER2 with a 39.2%, unlike the Asiatic indigenous population, where the most frequent was the luminal A type (33%). This correlates with greater risk and worse results in cancer among the indigenous communities.29\n\nFurthermore, OS decreased noticeably in stage IV, probably since cancer diagnosis in advanced stages is more biologically aggressive and presents high recurrence rates.30 Likewise, we found that NLR was associated with the disease staging, which could imply a confusing role of that variable when evaluating the predictive capacity of the NLR. Additionally, we found that the greater the age, the lower the OS. This finding is related to the study by Tao et al.31 where older patients with breast cancer had 17% greater mortality than younger patients. This age group also had a lower OS, since breast cancer at an older age may have late diagnosis and insufficient treatments.32\n\nBy studying the entire population of a referral hospital that accepts patients from all over the country, these results could be generalized to the urban context of Peru and other cities in Latin America, since the sociodemographic characteristics and the environment where the study was made is similar to that of other Latin American cities. On the other hand, although there was a long follow-up of eight years, during this period there was no important change in the treatment that significantly affected the survival of the patients evaluated, so the results are still useful.\n\nAmong the studyâs limitations, reporting bias likely exists due to the use of secondary sources such as medical records. Likewise, the patientsâ treatment was not reported, a covariable that could influence OS. However, patients were treated in the same hospital, therefore it is probable that they received the same treatment regimen directed towards their type of cancer.\n\n\nConclusions\n\nWe conclude that NLRâ¥3 is a risk factor for mortality, adjusted by age and clinical stage. Additionally, age and clinical stage IV could also be risk factors for mortality. A total of 59.3% of breast cancer patients had an NLRâ¥3, while the most frequently diagnosed stage was stage II with 42.74%, and the most frequent molecular subtype was HER2 with 39.2%.",
"appendix": "Data availability\n\nFigshare: Underlying data for âNeutrophil/lymphocyte ratio and overall survival in patients with breast cancer: a cohort study in a Latin-American hospitalâ, https://doi.org/10.6084/m9.figshare.20419401.v2. 33\n\nThe project contains the following underlying data:\n\nDATASET - Breast cancer - Hospital Hipolito Unanue (main data; in.xls and.dta format)\n\nFigshare: Underlying data for âNeutrophil/lymphocyte ratio and overall survival in patients with breast cancer: a cohort study in a Latin-American hospitalâ, https://doi.org/10.6084/m9.figshare.20419401.v2. 33\n\nThe project contains the following extended data:\n\n⢠Data collection sheet in English\n\n⢠Research protocol\n\nFigshare: STROBE checklist for âNeutrophil/lymphocyte ratio and overall survival in patients with breast cancer: a cohort study in a Latin-American hospitalâ, https://doi.org/10.6084/m9.figshare.20419401.v2. 33\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nWorld Health Organization: Breast cancer.2021 [cited 2022 Feb 1].Reference Source\n\nThun MJ, DeLancey JO, Center MM, et al.: The global burden of cancer: priorities for prevention. Carcinogenesis. 2010 Jan 1; 31(1): 100â110. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPan American Health Organization: Breast Cancer in the Americas.2014.Reference Source\n\nVallejos-Sologuren CS, Aguilar-Cartagena A, Flores-Flores CJ: Situación del Cáncer en el Perú. Diagnóstico. 2020 Nov 10; 59(2): 77â85. Publisher Full Text\n\nMaskarinec G, Pagano I, Lurie G, et al.: Factors Affecting Survival Among Women with Breast Cancer in Hawaii. J. Womens Health. 2011 Feb; 20(2): 231â237. Publisher Full Text\n\nSeedhom AE, Kamal NN: Factors Affecting Survival of Women Diagnosed with Breast Cancer in El-Minia Governorate, Egypt. Int J. Prev. Med. 2011 Mar 12 [cited 2022 Feb 1]; 2(3).Reference Source\n\nMinisterio de Salud del Perú: Análisis de la Situación del Cáncer en el Perú.2018. 2020 [cited 2022 Feb 1].Reference Source\n\nMinisterio de Salud del Perú: Plan nacional para la prevención y control de cáncer de mama en el Perú 2017-2021. Biblioteca Central del Ministerio de Salud;2017.Reference Source\n\nLynce F, Graves KD, Jandorf L, et al.: Genomic Disparities in Breast Cancer among Latinas. Cancer Control. 2016 Oct; 23(4): 359â372. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEnriquez D, De la Cruz Ku GA, Fernandez M, et al.: Predictive value of neutrophil-to-lymphocyte ratio on pathological complete response in triple negative breast cancer. J. Clin. Oncol. 2017 May 20; 35(15_suppl): e12012âe12012. Publisher Full Text\n\nde la Cruz-Ku G , Chambergo-Michilot D, Torres-Roman JS, et al.: Neutrophil-to-lymphocyte ratio predicts early mortality in females with metastatic triple-negative breast cancer. Coleman WB, editor. PLoS One. 2020 Dec 7; 15(12): e0243447. 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Cancer.Net.2012 [cited 2022 Feb 3].Reference Source\n\nBreastcancer.org: Análisis de inmunohistoquÃmica (IHQ).2015 [cited 2022 Feb 3].Reference SourceReference Source\n\nBuleje J, Guevara-Fujita M, Acosta O, et al.: Mutational analysis of BRCA1 and BRCA2 genes in Peruvian families with hereditary breast and ovarian cancer. Mol. Genet. Genomic Med. 2017 Sep; 5(5): 481â494. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarzan D, Veldwijk MR, Herskind C, et al.: Comparison of genetic variation of breast cancer susceptibility genes in Chinese and German populations. Eur. J. Hum. Genet. 2013 Nov; 21(11): 1286â1292. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinisterio de Salud del Perú, Instituto Nacional de Salud: Proyectos de investigación basados en las Prioridades Nacionales de Investigación: 2021 I. INSTITUTO NACIONAL DE SALUD;2021 [cited 2022 Feb 1].Reference Source\n\nEthier J-L, Desautels D, Templeton A, et al.: Prognostic role of neutrophil-to-lymphocyte ratio in breast cancer: a systematic review and meta-analysis. Breast Cancer Res. 2017 Dec; 19(1): 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Berckelaer C, Van Geyt M, Linders S, et al.: A high neutrophil-lymphocyte ratio and platelet-lymphocyte ratio are associated with a worse outcome in inflammatory breast cancer. Breast. 2020 Oct; 53: 212â220. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu L, Saxena S, Goel P, et al.: Breast Cancer CellâNeutrophil Interactions Enhance Neutrophil Survival and Pro-Tumorigenic Activities. Cancers. 2020 Oct 8; 12(10): 2884. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOcana A, Nieto-Jiménez C, Pandiella A, et al.: Neutrophils in cancer: prognostic role and therapeutic strategies. Mol. Cancer. 2017 Dec; 16(1): 137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVicente Conesa MA, Garcia-Martinez E, Gonzalez Billalabeitia E, et al.: Predictive value of peripheral blood lymphocyte count in breast cancer patients treated with primary chemotherapy. Breast. 2012 Aug; 21(4): 468â474. Publisher Full Text\n\nHirahara T, Arigami T, Yanagita S, et al.: Combined neutrophil-lymphocyte ratio and platelet-lymphocyte ratio predicts chemotherapy response and prognosis in patients with advanced gastric cancer. BMC Cancer. 2019 Dec; 19(1): 672. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrditura M, Galizia G, Diana A, et al.: Neutrophil to lymphocyte ratio (NLR) for prediction of distant metastasis-free survival (DMFS) in early breast cancer: a propensity score-matched analysis. ESMO Open. 2016; 1(2): e000038. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYersal O, ÃetinkÃŒnar S, Aktimur R, et al.: Neutrophil/Lymphocyte and Platelet/Lymphocyte Ratios are Not Different among Breast Cancer Subtypes. Asian Pac. J. Cancer Prev. 2017 Aug 1; 18(8): 2227â2231. PubMed Abstract\n\nElyasinia F, Keramati MR, Ahmadi F, et al.: Neutrophil-Lymphocyte Ratio in Different Stages of Breast Cancer. Acta Med. Iran. 2017 May 16; 55(4): 228â232.\n\nSegelov E, Garvey G: Cancer and Indigenous Populations: Time to End the Disparity. JCO Glob. Oncol. 2020 Nov; 6: 80â82. PubMed Abstract | Publisher Full Text\n\nGajdos C, Tartter PI, Bleiweiss IJ, et al.: Stage 0 to stage III breast cancer in young women11No competing interests declared. J. Am. Coll. Surg. 2000 May; 190(5): 523â529. Publisher Full Text\n\nTao L, Schwab RB, San Miguel Y, et al.: Breast Cancer Mortality in Older and Younger Patients in California. Cancer Epidemiol. Biomark. Prev. 2019 Feb; 28(2): 303â310. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTesarova P: Breast cancer in the elderlyâShould it be treated differently? Rep. Pract. Oncol. Radiother. 2013 Jan; 18(1): 26â33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOcaña MS, Quiñones Laveriano DM, De La Cruz-Vargas J, et al.:Dataset _ Neutrophil/lymphocyte ratio and overall survival in patients with breast cancer: A cohort study in a Latin-American hospital. [Dataset]. figshare. 2022. Publisher Full Text"
}
|
[
{
"id": "180426",
"date": "23 Nov 2023",
"name": "Helene Rundqvist",
"expertise": [
"Reviewer Expertise Breast cancer",
"immunology",
"hypoxia",
"exercise"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nContent\nThis article presents retrospective findings regarding the association of the neutrophil/lymphocyte ratio (NLR) with overall survival (OS) in 241 female breast cancer patients in Peru. Kaplan-Meier statistics and cox proportional hazard models were used to estimate risk of overall survival. Findings suggest an association between NLR and OS.\nGeneral Comments\nThe study population is of common interest as evidence from industrial countries may not be generalisable to cancer patients in developing countries. The study design is appropriate if the major revisions are addressed. There are sufficient details to allow replication. The statistical analysis and interpretation need to be revised, as there are several mistakes concerning data, results, and statistical testing. Data on participant characteristics are available but not the exposure variable. NLR and absolute neutrophil and lymphocyte counts should be added to the supplementary data file. Some conclusions are not properly drawn, and references need to be added to numerous statements.\nMajor Revisions\nThe study is a retrospective study, prospective needs to be changed to retrospective.\n\nThe article has already been shared on Research Gate, and it is not clearly stated that this publication has not been peer-reviewed. I would strongly suggest removing the article from research gate until the peer-review is completed (especially considering the major revision), or to clearly indicate that it has not been peer-reviewed.\nhttps://www.researchgate.net/publication/369519865_Neutrophillymphocyte_ratio_and_overall_survival_in_patients_with_breast_cancer_a_cohort_study_in_a_Latin-American_hospital\n\nThe lack of statistical significance regarding tumor subtype is likely due to the number of groups and the small number of observations. Pathologically, it is expected that tumor subtypes affects both NLR and survival. Therefore, I strongly believe that subtypes need to be added to the multivariable model as a confounder, irrespectively of univariable statistical significance.\n\nThe publication is lacking sensitivity analyses:\nI strongly encourage statistical comparison between baseline characteristics between groups, especially mean ±SD of continuous age, and an addition of mean ± SD of raw neutrophil and leukocyte counts.\n\nIt could be of benefit to add a sensitivity analysis with removal of overall mortality events six months after baseline to remove mortality that the individuals were predisposed to before cancer diagnosis.\n\nMoreover, survival models for raw lymphocyte and neutrophil counts may be added.\n\nP-values in Figure 2 differ from p-values reported in text:\n\nFigure 2C and 2D both include p =0.017\n\nâWhen evaluating OS by molecular subtype, no statistically significant differences are observed (p=0.528) (Figure 2B). P-values differ from Figure 2B (p=0,444).\n\nI suggest statistical testing for p-values in figures and tables to be added to the legend text.\n\nHRa at 68 for Stage 4 in abstract needs to be corrected.\n\nAs age is available as continuous variable, there is no reason to dichotomize the variable. Since the age range is quite high (26-85), a binary variable is not enough to adjust away differences of mortality risk across age. Moreover, the cut-off at 55 is not elaborated on in the method section.\n\nIn the text you state:\nâTotal of 59.3% of breast cancer patients had an NLRâ¥3â\n\nâFurthermore, 144 (59.8%) patients had NLRâ¥3.â â\n\nâI found that over half of patients had an NLRâ¥3, similar to that found in other studies.â\n\nHowever, in the Table 1 AND 2 the numbers are NLR<3 = 144 and NLRâ¥3=97. This is a very critical mistake. Please correct throughout manuscript.\nYou state: âIn our study, I found that NLRâ¥3 (p<0.001), age>55 years (p=0.039), and clinical stage (p<0.001), were associated with a lower OS.â â Where does the p-value 0.039 come from? It is not reported in any table or figure, and it needs to be clarified what the p-values refer to.\n\nTable 3 â I understand the bivariate refers to models for NLR and one independent variable and the multivariate refers to models for NLR and significant confounders from univariable testing? Bivariate and multivariate needs to be changed to univariable and multivariable  as the ending -variate refers to response variables whereas -variable refers to independent variables, i.e. see link. Moreover, please add information about confounders included in multivariable models to the table legend.\n\nSuggest to rephase âWe hope to findâ to âWe hypothesized that there is an association âŠâ in introduction.\nMinor Revisions\nPlease change term âqualitativeâ variables to âcategoricalâ or âdiscreteâ variables (see link)\n\nThere are several parts that require references to previous literature.\nâWhile research exists on an international level that associates an elevated NLR with lower OS in patients with breast cancer, these were developed in populations with early-stage diagnosis, as opposed to countries with middle and low incomes, where a high proportion are diagnosed in advanced stages.â\n\nâSimilar results were found by a Peruvian study, only in breast cancer with a triple-negative molecular subtype, while in our study I evidenced the use of this ratio in other molecular subtypes.â\n\nâOn the other hand, a decrease in lymphocyte count may decrease the effectiveness of the anti-tumoral immune responseâ\n\nâBreast cancer mortality after 5 years could be high due to diverse factors foreign to what NLR might reflect, such as tumoral growth, comorbidities, and age-related functional impairmentâ\n\nâThe most frequent molecular subtype was HER2 with a 39.2%, unlike the Asiatic indigenous population, where the most frequent was the luminal A type (33%).â\n\nPlease elaborate on how excluded observations were handled, i.e., considering Table 1.\n\nPlease comment on the high incidence of HER2 in the population in the discussion. Does this reflect trends in South America, please refer to current literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-331
|
https://f1000research.com/articles/12-136/v1
|
06 Feb 23
|
{
"type": "Research Article",
"title": "Ocimum sanctum Linn. Ethanolic extract promotes an antiproliferative and apoptosis activity in MCF-7 and T47D breast cancer cell lines mediated by upregulation of ROS/RNS, Caspase 9, and Caspase 3: an in silico and in vitro study",
"authors": [
"Hevi Wihadmadyatami",
"Srikanth Karnati",
"Suleyman Ergún",
"Ulayatul Kustiati",
"Dewi Ratih Tirtosari",
"Dwi Liliek Kusindarta",
"Yudy Tjahjono",
"Srikanth Karnati",
"Suleyman Ergún",
"Ulayatul Kustiati",
"Dewi Ratih Tirtosari",
"Dwi Liliek Kusindarta",
"Yudy Tjahjono"
],
"abstract": "Breast cancer is the most serious disease affecting women worldwide. Recently, breast cancer cases reached 2.2 million people. The treatment method is still developing. In addition, the use of herbal medicine as a palliative therapeutic to chemical and/or synthetic drugs is increasing. Ocimum sanctum Linn. is a popular plant in Indonesia and Southeast Asia countries and is also known as an herbal medicinal plant. The study aimed to prove the ability of ethanolic extract Ocimum sanctum Linn. (EEOS) as an antiproliferative against breast cancer. Cytotoxic assay, adhesion assay, and Reactive Nitrogen Species (RNS) production determined in MCF-7 and T47D breast cancer cell. Furthermore, SEM is applied to visualize cell morphology. In addition, molecular docking is also performed. The result shows EEOS inhibited the proliferation and adhesion of the MCF7 and T47D cells line. Surface morphology showed that MCF7 and T47D tend to be apoptotic (cells turned rougher, gritty, and blebbing). EEOS also increased RNS production. Molecular docking describes the phytochemical compounds on the EEOS (gallic acid, caffeic acid, rosmarinic acid and apigenin) interacted with the caspase-3 and caspase-9. In conclusion, EEOS can inhibit the proliferation of MCF-7 and T47D breast cancer cell lines that correlate with upregulated RNS production, as well as the expression of Caspase 3 and Caspase 9.",
"keywords": [
"Ethanolic Extract Ocimum sanctum Lin",
"Breast Cancer",
"RNS",
"Insilico molecular docking",
"An-ti-proliferative",
"Apoptosis",
"Caspase 3",
"Caspase 9"
],
"content": "Introduction\n\nCancer is becoming more common globally. Breast cancer is a serious illness and the leading cause of death, accounting for 23% of all cancers and 14% of cancer-related deaths in women worldwide.1 Increasing age, reproductive, and genetic factors are known risk factors for breast.2 Breast cancer is a common female malignancy, affecting 2.1 million women annually.3 Surgery, radiotherapy, chemotherapy, endocrine therapy, and targeted therapy are breast cancer treatment strategies. Chemotherapy plays an important role in breast cancer treatment.4 It is widely used in combination with surgery and radiation therapy, although the efficacy of this treatment is still the leading cause of death.5 Chemotherapy, as a cancer treatment, uses cytotoxic agents to kill cancer cells. This agent destroys tumor tissue and cannot distinguish normal cells from cancer, moreover, the method is not effective if cancer already undergoes metastases. Therefore, new strategies to effectively control cancer growth are urgently needed.6 While modern and conventional medicine has been used as the main medicine for cancer treatment, traditional herbal medicine has been commonly used as a complementary and alternative strategy in several countries.7\n\nPlants have long been known for their therapeutic properties. Herbal products can be trusted for cancer treatment because of their low toxicity. Most women with breast cancer easily accept herbal medicines because of their availability, affordable prices, ease of implementation, and acceptance to control cancer cell growth.8 In these relations, it is important to mention that Ocimum sanctum Linn. (OS) is an herbal plant native to Asian countries that has several beneficial biological activities,9 especially anti-inflammatory10 and antioxidant activity.11 However, relatively few studies have investigated the biological activity underlying the cytotoxic effect of EEOS treatment in altering the survival of lung adenocarcinoma A549 cells through the synergistic induction of apoptosis signaling via the intrinsic mitochondrial pathway,12 the ethanolic extract of OS inhibited A549 cell angiogenesis by how to reduce the expression of αvβ3 integrins, MMP-2, and MMP-9.13 To provide scientific justification for the use of herbal medicines, it is necessary to record and publish preclinical evidence and clinical-based research. Therefore, this study was designed to analyze the ability of the ethanolic extract of O. sanctum Linn leaves as an anticancer agent in breast cancer cells, mainly to prove the ability to inhibit the proliferation of cancer cells.\n\n\nMethods\n\nThe leaves of Ocimum sanctum Linn simplisia were obtained from center of agrotechnology, Universitas Gadjah Mada and the species identified at the department of biology, Universitas Gadjah Mada (Yogyakarta, Indonesia). The ethanolic extract was obtained by maceration technique.12 A total of 4000 ml of 96% ethanol (Merck, Darmstadt, Germany) was added to 300 grams simplicia Ocimum sanctum Linn. The filtration results concentrated using a vacuum rotary evaporator (Heidolph, Schwabach, Germany), and 8.82% w/w of ethanol extract of Ocimum sanctum Linn was obtained in the form of a paste.\n\nMCF-7 and T47D cells were cultivated in T25 flasks with Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Langenselbold, Germany) supplemented with Fetal Bovine Serum (FBS) (Capricorn, Ebsdorfergrund, Germany) 10%, Penicillin-Streptomycin (Capricorn, Ebsdorfergrund, Germany) 2%, amphotericin B 0.5%, and Non-Essential Amino Acids (NEAA) 1%. The T25 flask was then stored in an incubator at 37°C and 5% CO2. T47D cells were cultured in T25 flasks with Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, Langenselbold, Germany) supplemented with Fetal Bovine Serum (Capricorn, Ebsdorfergrund, Germany) 10%, penicillin-streptomycin (Capricorn, Ebsdorfergrund, Germany) 2%, and 0.5% amphotericin-B, and stored in an incubator at 37°C and 5% CO2.\n\nCytotoxicity ability of EEOS examined by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). 80% confluence cell of MCF-7 and T47D breast cells were grown on a 96-well culture test plate and incubated at 37°C and 5% CO2 for 24 hours. Cell cultures were rinsed with Dulbecco's Phosphate Buffer Saline (DPBS) (Capricorn, Ebsdorfergrund, Germany) prior to treatment. Cells were divided into untreated (NT), cisplatin (Kalbe, Jakarta, Indonesia) 35 ÎŒg/ml (MCF-7) and 50 ÎŒg/ml (T47D cells) groups as positive controls with commercial drugs for the treatment of breast cancer, and EEOS with graded concentrations of 50, 100, 150, 200, and 250 ÎŒg/ml (MCF-7) and 50, 75, 150, 225, and 300 ÎŒg/ml (T47D cells). The analysis was run as triplicate. The treated cells were incubated at 37°C and 5% CO2 for 24 hours. The media was removed and rinsed with DPBS, then 0.5 mg/ml MTT reagent was added to 100 l/well. Cells were incubated for 4 hours and then given Dimethyl sulfoxide (Sigma-Aldrich, Munich, Germany) 100 ÎŒl/well to stop the formation of formazan crystals. The optical density value was read using a microplate reader (Tecan, Zurich, Switzerland) with a wavelength of 595 nm. The results of the absorbance optical density value are calculated using the formula:\n\nThe ability of EEOS to inhibit the adhesion of breast cancer cells was examined by the adhesion test using the CCK-8 test (Cell Counting Kit-8) (Abbkine cat: KTC011001-3, California, USA) with Water Soluble Tetrazolium (WST)-8 reagent. MCF-7 and T47D breast cancer cells were grown on 96 well culture test plates and incubated at 37°C and 5% CO2 for 24 h. Cells were then treated by EEOS. After being treated and incubated for 24 h, the media was removed and rinsed with DPBS, then 100 l of 10% WST-8 reagent was added. Cells were incubated for 1 h. The optical density value was read using a microplate reader (Tecan, Zurich, Switzerland) with a wavelength of 450 nm. The results of the absorbance optical density value are calculated using the formula:\n\nConfluent MCF-7 and T47D breast cancer cells were grown on 6-well culture test plates and then incubated at 37°C and 5% CO2 for 24 h. Cells were washed with DPBS and then treated with EEOS after incubated for 24 hours, rinsed the cultivation with DPBS and lysed using 1000 ÎŒl of RIPA lysis buffer. The adhering cells were removed with the aid of a cell scraper and then transferred to micro-tubes and centrifuged at 6500 rpm for 10 min at 4°C. The resulting supernatant was taken for the Griess test.\n\nThe MCF-7 and T47D cell lysates supernatants (100 ÎŒl) and standard solutions were transferred to 96 wells assay plate culture. Add 0.1% N-1-naphtyletthylenediamine dihydrochloride (NEDD) and 2% sulfanilamide in 5% HCl 50 l each to each well simultaneously. The absorbance value was read by a microplate reader with a wavelength (λ) of 540 nm. The average value of the absorbance was compared with the standard value of nitride oxide (NO) to obtain the NO concentration.\n\nInterventionary studies involving animals or humans, and other studies that require ethical approval, must list the authority that provided approval and the corresponding ethical approval code.\n\nConfluent MCF-7 and T47D breast cancer cells were grown on a 24-well culture test plate, covered with a sterile coverslip and allowed for 30 minutes, then added 200 ÎŒl of media and incubated at 37°C and 5% CO2. for 24 h. Cells were then treated with EEOS and carried out in duplicate. Cells were rinsed with DPBS and fixed using 250 ÎŒl of 2.5% glutaraldehyde, cells were incubated for 2 hours at 4°C. The slides were then dehydrated using a series of graded ethanol solutions (20, 30, 40, 50, 60, 70, and 100%). The specimens were vacuum dried (25°C, 4 Pa; Buehler 1000 Vacuum System, Stuttgart, Germany) and coated with platinum (JEOL JEC-3000FC, Tokyo, Japan), then examined under an SEM (JEOL-JSM6510LA, Tokyo, Japan) using a 15-kV acceleration voltage.\n\nThe four bioactive compounds of the EEOS were identified as apigenin (CID_5280443),43 caffeic acid (CID_689043),44 and Gallic acid (CID_370),45 dan rosmarinic acid (CID_5281792),46 downloaded from the PubChem database (see Data availability).\n\nThe target proteins used in this in silico study were caspase-9 (PDB ID:1jxq)47 and caspase-3 (PDB ID: 1nms),48 which are downloaded from the structural information PDB.\n\nBioactive compounds (apigenin, gallic acid, caffeic acid, and rosmarinic acid) and target proteins (caspase-9 and caspase-3) were prepared sequentially using the Discovery Studio v. 19.0.0 program and the PyRX 0.8 program. The proteins and bioactive compounds were interacted with the HEX 8.0.0 program and visualized with the Discovery Studio Visualizer v. 19.0.0.\n\nStatistical performed using one-way analysis of variance followed by the appropriate post hoc Tukey test, p<0.05 was assumed to represent statistical significance. Statistical analysis was run using GraphPad Prism 7.0 (La Jolla, CA, USA).\n\n\nResults\n\nMCF-7 and T47D cells were cultured on well plates and treated for 24 hours with different concentrations of EEOS. MCF-7 cells are treated with EEOS in 50, 100, 150, 200, and 250 ÎŒg/ml concentrations. In another hand, T47D cells are treated with 75, 150, 225, 300, and 375 ÎŒg/ml EEOS. Cisplatin is given as positive control as a comparison to commercial drugs. EEOS induced cell death and inhibit cell attachment in MCF-7 cell dose-dependent manner (Figures 1A, 2A) the optimal concentration of EEOS, 100 ÎŒg/ml, showed a little amount of attaching cell in comparison with other concentrations (Figure 1). Furthermore, EEOS also induced cell death and inhibit cell attachment, but non-significant in 75 and 150 ÎŒg/ml concentration of EEOS (Figures 1B, 2B). The optimum concentration EEOS to inhibit the growth of T47D cells is 375 ÎŒg/ml and showed enormous cell detachment with others.\n\n(EEOS) against MCF-7 (A) and T47D (B) breast cancer cells. MCF-7 breast cancer cells (A) were cultivated in the presence of cisplatin 35 ÎŒg/ml as the commercial drug comparison, and EEOS at concentrations of 50, 100, 150, 200, dan 250 ÎŒg/ml. T47D breast cancer cells (B) were cultivated in the presence of cisplatin 50 ÎŒg/ml as the commercial drug comparison, and EEOS at concentrations of 75, 150, 225, 300, and 375 ÎŒg/ml. After 24 h, EEOSâs inhibitory effect was visualized by an MTT reagent at a wavelength of 595 nm (NT: Non-treated; **significant p<0.0062; ****significant p<0.0001; n.s.=Not significant).\n\n(EEOS) against MCF-7 (A) and T47D (B) breast cancer cells. MCF-7 breast cancer cells (A) were cultivated in the presence of cisplatin 35 ÎŒg/ml as the commercial drug comparison, and EEOS at concentrations of 50, 100, 150, 200, dan 250 ÎŒg/ml. T47D breast cancer cells (B) were cultivated in the presence of cisplatin 50 ÎŒg/ml as the commercial drug comparison, and EEOS at concentrations of 75, 150, 225, 300, and 375 ÎŒg/ml. After 24 h, EEOSâs inhibitory effect was visualized by an MTT reagent at a wavelength of 595 nm (NT:Non-treated; **significant p<0.0062; ****significant p<0.0001; n.s.=Not significant).\n\nIn addition, SEM was performed to visualize the morphological surface of MCF-7 (Figure 3) and T47D (Figure 4) breast cancer cells. In the administration of EEOS with gradual concentrations on MCF-7 cells: 50 ÎŒg/ml EEOS (Figure 3C), 100 ÎŒg/ml EEOS (Figure 3D), 150 ÎŒg/ml EEOS (Figure 3E), 200 ÎŒg/ml EEOS (Figure 3F), and 250 ÎŒg/ml (Figure 3G), a protrusion appeared on the cell surface followed by the formation of holes on the cell surface, where more holes appeared as the concentration of EEOS was increased. In addition, the experiment was also performed In T47D cells treated in the presence of EEOS at gradual concentrations. Several concentrations of EEOS are 50 ÎŒg/ml EEOS (Figure 4C), 75 ÎŒg/ml EEOS (Figure 4D), 150 ÎŒg/ml EEOS (Figure 4E), 225 ÎŒg/ml EEOS (Figure 4F), and 300 ÎŒg/ml EEOS (Figure 4G). In the treatment of EEOS, the T47D showed quite clear changes; the cells looked more gritty, with holes and blebbing on the surface of the cells; at the highest concentration (300 ÎŒg/ml), the cells appeared flattened and more holes (Figure 4). Morphological changes in both cell lines, the appearance of roughness, holes, or blebbing, are markers that MCF-7 and T47D cells undergo apoptosis. These results are also consistent with the surface structure of MCF-7 cells (Figure 3B) and T47D (Figure 4B) with the administration of cisplatin as one of the commercial drugs used to treat breast cancer today.\n\n(EEOS) A) Non treated cells, in the morphologycal analysis of the cells show no cells membrane blebing and hole on the cell surface, (B) MCF-7 cells in the presence of Cisplatin 35 ÎŒg/ml, on the surface of the cells, the cells blebing are appear, in addittion the same characteristic of necrotic or apoptotic cells also found in MCF-7 in the presence 50 ÎŒg/ml EEOS (C), and furthermore in the treatment of 100 ÎŒg/ml EEOS (D), 150 ÎŒg/ml EEOS (E), treatment with 200 ÎŒg/ml EEOS (F), and treatment with 250 ÎŒg/ml (G) beside the blebbing, the cells have more hole or pit on the surface which indicate the cells undergo to apoptosis.\n\n(EEOS) A) Non treated cells, in the morphologycal analysis of the cells show no cells membrane blebing and hole on the cell surface, (B) T47D cells in the presence of Cisplatin 35 ÎŒg/ml, on the surface of the cells, the cells hole are appear, in addittion the same characteristic of necrotic or apoptotic cells also found in T47D in the presence 50 ÎŒg/ml EEOS there is some blebbing and hole in the surface of the cells(C), meanwhile on 75 ÎŒg/ml EEOS the holes more appear on the surface(D), furthermore in the treatment of 150 ÎŒg/ml EEOS (E), 225 ÎŒg/ml EEOS (F), and treatment with 300 ÎŒg/ml (G) the cells have more hole or pit as well as blebbing on the surface. In addition in the presence of 300 ÎŒg/ml (G) the cells describe more flattened with blebbing and hole in compare to non trated cells (A). The changes of cells morphology indicate the cells undergo to apoptosis.\n\nTo demonstrate the ability of EEOS to induce an apoptotic effect on MCF-7 and T47D breast cancer cells we performed Griess assay to measure the concentration of Nitric Oxide (NO) as a part of Reactive Nitrogen Species (RNS). Indeed, this experiment showed that concentration of NO in MCF-7 and T4TD breast cancer cells were increased in line with increase in EEOS concentration (Figure 5).\n\nMCF-7 breast cancer cells (A) were cultivated in the presence of cisplatin 35 ÎŒg/ml as the commercial drug comparison, and EEOS at concentrations of 50, 100, 150, 200, dan 250 ÎŒg/ml. T47D breast cancer cells (B) were cultivated in the presence of cisplatin 50 ÎŒg/ml as the commercial drug comparison, and EEOS at concentrations of 75, 150, 225, 300, and 375 ÎŒg/ml. After 24 h, EEOSâs inhibitory effect was visualized by an MTT reagent at a wavelength of 595 nm (NT:Non-treated; **significant p<0.0062; ****significant p<0.0001; n.s.=Not significant).\n\nThree amino acid residues of protein caspase 3 bound to gallic acid in THR166, GLU123, and GLU167 with an energy of -167.9 kJ/mol (Figure 6, Table 1). Gallic acid compounds showed different interaction results when docked with caspase 9, two amino acid residues (SER194, GLU161) were found that interacted with and bond energy of -101.9 kJ/mol (Figure 7, Table 1).\n\n(A) Three-dimensional structural interaction between the ligand Gallic Acid (blue) and caspase 3 (yellow). This interaction between Gallic Acid on the active site of caspase 3, called THR166, GLU123, GLU167, which can be seen in the two-dimensional (B) and three-dimensional structures (C).\n\n(A) Three-dimensional structural interaction between the ligand Gallic Acid (blue) and caspase 9 (green). This interaction between Gallic Acid on the active site of caspase 9, called, SER194, GLU161, which can be seen in the two-dimensional (B) and three-dimensional structures (C).\n\nCaffeic acid compounds and protein caspase 3 binds to amino acid residues ASN35, ARG241, and THR245, this interaction forms affinity energy of -214.4kJ/mol (Figure 8, Table 2). Caffeic acid compounds that interacted with protein caspase 9 showed activity with total energy -117.0kJ/mol, the interaction found that there was one amino acid residue that interacted with caffeic acid, namely GLU161 (Figure 9, Table 2).\n\n(A) Three-dimensional structural interaction between the ligand Caffeic Acid (blue) and caspase 3 (yellow). This interaction between Caffeic Acid on the active site of caspase 3, called, ASN35, ARG241, THR245, which can be seen in the two-dimensional (B) and three-dimensional structures (C).\n\n(A) Three-dimensional structural interaction between the ligand Caffeic Acid (blue) and caspase 9 (green). This interaction between Caffeic Acid on the active site of caspase 9, called, GLU161, which can be seen in the two-dimensional (B) and three-dimensional structures (C).\n\nRosmarinic acid docked with protein caspase 3 forms an energy affinity of -265.8 kJ/mol. This bond indicates the presence of amino acid residues that contribute to binding rosmarinic acid compounds, namely ARG164, CYS264, GLY165, GLU124, PRO201, VAL266, LYS137 (Figure 10, Table 3). Rosmarinic acid binds caspase 9 at the amino acid residue ARG341 have an energy of -180 kJ/mol (Figure 11, Table 3).\n\n(A) Three-dimensional structural interaction between the ligand Rosmarinic Acid (blue) and caspase 3 (yellow). This interaction between Rosmarinic Acid on the active site of caspase 3, called, ARG164, CYS264, GLY165, GLU124, PRO201, VAL266, LYS137, which can be seen in the two-dimensional (B) and three-dimensional structures (C).\n\n(A) Three-dimensional structural interaction between the ligand Rosmarinic Acid (blue) and caspase 9 (green). This interaction between Rosmarinic Acid on the active site of caspase 9, called, ARG341, which can be seen in the two-dimensional (B) and three-dimensional structures (C).\n\nInteraction results between caspase 3 and Apigenin show there are three amino acid residues binding apigenin namely PHE247, TRP241, dan LYS242 with energy -245.3 kJ/mol (Figure 12, Table 4). Meanwhile, there is no binding between caspase 9 and apigenin, but still produces energy -145.2 kJ/mol (Figure 13, Table 4).\n\n(A) Three-dimensional structural interaction between the ligand Apigenin (blue) and caspase 3 (yellow). This interaction between Apigenin on the active site of caspase 3, called, PHE247, TRP241, dan LYS242, which can be seen in the two-dimensional (B) and three-dimensional structures (C).\n\n(A) Three-dimensional structural interaction between the ligand Apigenin (blue) and caspase 9 (green). There is no interaction between Apigenin on the active site of caspase 9, which can be seen in the two-dimensional (B) and three-dimensional structures (C).\n\n\nDiscussion\n\nCurrently, breast cancer treatment requires collaborative efforts among several treatment methods. Chemotherapy shows extraordinary efficacy in destroying cancer cells but causes side effects, such as nausea and vomiting, diarrhea, constipation, and alopecia, to impaired immune function. Side effects' severity depends on the drug type, dosage, duration of treatment, and the patient's condition.14 For centuries, herbal plants and their extracts have been used as food and medicine. This review concerns various plants that retain their anti-tumour properties.15 Therefore, herbal medicines are popular, so scientific studies are needed to prove them. It has been reported that Ocimum sanctum is an herbal plant with many health benefits. Studies have revealed the potential of EEOS as an anticancer drug candidate. EEOS can induce apoptotic activity through the mitochondrial pathway12 and inhibit angiogenesis of the human lung adenocarcinoma cell line A549,13 EEOS inhibits invasion and reduces the activity of MMP-2 and MMP-9 HN4 and carcinoma head and neck squamous cells HN12,16 EEOS has cytotoxic ability against leukemia cells K56217 and oral cancer cell line Ca9-22.18\n\nIn this study, EEOS demonstrated a significant antiproliferative effect against the MCF-7 and T47D human breast carcinoma cell lines. The MTT assay evaluated the cytotoxic efficacy of O. sanctum. The results showed that the number of surviving MCF-7 cells was significantly lower when treated with various forms of extract compared to the control (Figure 1A). On the other hand, T47D cell viability decreased significantly at a concentration of 375 (ÎŒg/ml) (Figure 1B). When the dose-dependent effect was evaluated, all groups showed a steady decrease in the percentage of surviving cells. The ability of EEOS to inhibit the adhesion of MCF-7 and T47D cells were tested using the CCK-8 assay and it was seen that the viability of MCF-7 and T47D cells decreased in line with increasing EEOS concentration (Figure 2A, B). This finding could be interpreted as increasing the concentration of EEOS, the cytotoxic activity and adhesion of the extract increased stably with a significant correlation.\n\nMorphological evaluation of breast cancer cell lines treated with EEOS using SEM revealed the positive cytotoxic ability of EEOS. The morphological changes of the two cell lines, which are characteristic of apoptosis, showed the anticancer activity of O. sanctum leaves with ethanolic extract (Figures 3, 4). According to Yulianto et al. (2017),19 the morphology of cells given anticancer compounds will experience (cell shrinkage) and become denser, and cell organelles will be more tightly packed. Haryanti and Widi-yastuti (2017)20 added that changes in cell morphology in the form of differences in cell dimensions (shrinkage), cytoplasmic compaction, and extracellular matrix damage indicate the characteristics of cells undergoing apoptosis. To clarify the results obtained, we measured the levels of NO) with the Griess test. NO is a ubiquitous water-soluble gas with free radicals. NO also plays a role in inflammatory and immune responses. However, the role of NO in tumors needs to explore deeply. A recent study conducted on the NO has been shown to have anticancer effects. These effects depend on the time, location, and concentration of NO in cells.21 This study showed that on incubation of MCF 7 and T47D cells treated with EEOS, produces higher the NO in line with higher EEOS concentration (Figure 5). NO has been studied as a potential promoter of breast cancer, and increased concentrations of NO were detected in the blood of breast cancer patients.22 An increase in the amount of NO was observed in MCF-7 cells treated with the methanol extract of Fraxinus micrantha.23 NO is produced in a concentration and time-dependent manner. The expression of several genes involved in tumor biology and tumor development is regulated by NO and is largely regulated by NO-mediated posttranslational modification (PTM) protein. The affected proteins cause cellular dysfunction that contributes to cancer onset, growth, development, invasion, and metastasis.24\n\nKumar and Kashyap (2015)23 stated that an increase in NO levels could occur due to the administration of various agents that cause apoptosis. A wide variety of active phytochemicals, such as flavonoids, phenols, terpenoids, saponins, alkaloids, steroids, and tannins have been identified in EEOS.25 EEOS phytochemical compounds can act as antiproliferative and apoptotic agents in NSCLC cells by in silico molecular docking and in vitro experiments with different mechanisms of action.26 Studies showed that the phytochemicals, quercetin, and genistein (flavonoid members), epigallocatechin-3-gallate (phenol members), and sulforaphane induce NO production, alter the redox environment, and thereby facilitate apoptosis. NO is produced by three NOS enzymes. The mRNA transcript levels of the three NOS enzymes after phytochemical treatment were assessed, and it was shown that after treatment with all phytochemicals, the expression of two or more NOS transcripts increased. Furthermore, the protein levels of iNOS and eNOS were found to be significantly increased.27\n\nIn former research, the ethanolic extract of Ocimum sanctum Linn. contains active compounds of flavonoids, phenols, alkaloids, tannins, saponins, terpenoids, and steroids.28 The active compounds in plants have anticancer effects and are used as therapeutic agents with low toxicity. In this study, Gallic acid (GA) is a natural phenolic compound that has a synergistic anticancer effect with paclitaxel and carboplatin to reduce proliferation. These effects are mediated through inhibition of the mitotic cycle, increased apoptosis, and overexpression of P53, Bax, and caspase-3 in breast cancer29 and cervical cancer.30 Caffeic acid (CA) is an antioxidant in normal cells, and pro-oxidant cells in tumor cells have low toxicity in normal cells and can inhibit cancer growth. Caffeic acid can suppress cell proliferation in breast cancer by mimicking antiestrogen activity and regulating growth regulators Estrogen Receptor (ER)/cyclin D1 and Insulin-Like Growth Factor I Receptor (IGF-IR)/pAkt, thereby leading to the development of cell cycle progression of damaged cells and decreased cell proliferation (Rosendahl et al., 2015), re-ducing IL-2 and activating NF-κB (Chen et al., 2018) and apoptotic ability through the intrinsic mitochondrial pathway.31,32\n\nRosmarinic acid (RA) also has phenol bioactive properties as an anticancer. RA inhibits cell proliferation and induces apoptosis in breast cancer and cervical cancer through cell cycle studies and apoptosis33 An article shows that RA has the ability as an anti-inflammatory and antioxidant. As an anticancer, RA can inhibit cell proliferation and migration, induce apoptosis, and as antiangiogenesis, it is expected to prevent tumor growth and metastasis.34 Research by Anwar et al., (2020)35 showed that atomistically, RA can inhibit microtubule regulatory kinase (MARK4) from controlling cell growth and inducing apoptosis. Apigenin, a natural bioflavonoid, is reported having low cytotoxicity and precise anticancer properties. Apigenin regulates signaling and participates in cancer cell metastasis, proliferation, and invasion.36 Apigenin induces apoptosis via an extrinsic caspase-dependent pathway in breast cancer37 and decreases CK2α expression levels in cervical cancer.38 Apigenin is able to induce ROS in MCF-7 intracellular cells thereby triggering the activation of p21 activation to induce termination of G2/M cells by promoting Cdc25C and CDK/cyclin. Moreover, ROS induction is also responsible for p53 activation, followed by activation of the caspase cascade pathway.39 Apigenin also has antiproliferative effects via estrogen receptor signalling and Akt/FOXO3a/FOXM1 signalling.40\n\nIn this current study, we used in silico molecular docking approaches to determine the mechanism of action of the EEOS. In cancer, there is an imbalance between cell differentiation and cell apoptosis. Apoptosis has an important role to maintain cellular homeostasis. In the process of apoptosis, caspases have a role in initiation and execution. Interestingly, in this study, we found a novelty in which we found other active sites of caspase bound by compounds of natural origin, namely ASN35, ARG241, THR245, THR166, GLU123, GLU167, PHE247, TRP214, LYS242, ARG164, CYS264, GLY165, GLU124, PRO201, VAL266, LYS137 with hydrogen bonds, Pi-Anion, Pi-Alkyl, Pi-Pi T-shaped, and Pi-Cation. Binding to the active site of caspase 3 affects the activation of caspase 3 and its regulation as a stimulator of apoptosis in cells. On the other hand, the amino acids GLU161, SER194, and ARG341 also bind to the active site of caspase 9 by Pi-Anion, Pi-Alkyl, and hydrogen bonds (Figure 14). In the process of apoptosis, the executor caspase will be activated when cleavage occurs by upstream initiators (including caspase 9) or additional proteases. Caspase 9 has a caspase recruitment domain (CARD)âapoptotic peptidase activating factor 1 (APAF1) apoptosome for caspase-9.41 Apoptosomes recruit and activate caspase-9, which continues the caspase cascade by cleaving caspases-3 and -7. To cleave caspase-3, caspase-9 needs to remain bound to the apoptosome and caspase-9 autocleavage to the p35/p17 subunit induces dissociation from apoptosis. Caspase-9 or caspase-3 in cancer is involved in apoptosis downstream of mitochondrial outer membrane permeabilization (MOMP). These caspase-9 and caspase-3-based therapies can induce MOMP and increase their cytotoxicity.42 Our study demonstrates that EEOS inhibits proliferation of MCF-7 and T47D breast cell cancer by increasing production of Reactive Nitrogen Species (RNS), thus altering the morphology of cells, and ending up with the increasing activity of Caspase-3 and Caspase 9. Further studies to know more deeply the mechanisms of how ethanolic extract Ocimum sanctum Linn. exerts its anticancer activity are required.\n\ntriggers the increasing expression of RNS/ROS in addition the phytochemical also interacts with the active site of Caspase 9 and Caspase 3 thus may increase the expression of Caspase 3 as well as Caspase 9 and push the cells going to apoptosis.\n\nThis study did not use material from animals or humans as test samples, thus ethical approval was not required.",
"appendix": "Data availability\n\nPubChem: Apigenin. Accession number: 5280443, https://identifiers.org/pubchem.compound:5280443. 43\n\nPubChem: Caffeic Acid. Accession number: 689043, https://identifiers.org/pubchem.compound:689043. 44\n\nPubChem: Gallic Acid. Accession number: 370, https://identifiers.org/pubchem.compound:370. 45\n\nPubChem: Rosmarinic acid. Accession number: 5281792, https://identifiers.org/pubchem.compound:5281792. 46\n\nPBD: Structure of cleaved, CARD domain deleted Caspase-9. Accession number: 1jxq, https://identifiers.org/pdb:1jxq. 47\n\nPBD: Caspase-3 tethered to irreversible inhibitor. Accession number: 1nms, https://identifiers.org/pdb:1nms. 48\n\n\nAcknowledgments\n\nThe authors wish to thank World Class Professor Program 2022, for the facilitation of fine tuning during the preparation of the manuscript. We thank to Integrated Laboratory for Research and Testing for the use of Scanning Electron Microscope, and also Yusuf Umardani, M.Eng., Puspa Hening, M. Biotech, Golda Rani Saragih, D.V.M. for excellent technical assistance.\n\n\nReferences\n\nTorre LA, Islami F, Siegel RL, et al.: Global Cancer in Women: Burden and Trends. Cancer Epidemiol. Biomark. Prev. 2017; 26: 444â457. Publisher Full Text\n\nKori S: An Overview: Several Causes of Breast Cancer. Epidemol. Int. J. 2018; 2(1): 01â17. Publisher Full Text\n\nMomenimovahed Z, Salehiniya H: Epidemiological Characteristics of and Risk Factors for Breast Cancer in the World. Breast Cancer. 2019; 11: 151â164. PubMed Abstract | Publisher Full Text\n\nWaks AG, Winer EP: Breast Cancer Treatment: A Review. JAMA - J. Am. Med. Assoc. 2019; 321: 288â300. Publisher Full Text\n\nGuan X: Cancer Metastases: Challenges and Opportunities. Acta Pharm. Sin. B. 2015; 5: 402â418. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou Z, Peng Y, Ai W, et al.: Compound Opening Arrow Mixture Exerts Anti-Tumor Effects in a Mouse Model of Breast Cancer. Sci. Rep. 2020; 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbu Bakar MF, Saifudin A, Cao P, et al.: Herbal Medicine for Prevention and Therapy in Breast Cancer. Evid. Based Complement. Alternat. Med. 2021; 2021: 1â4. Publisher Full Text\n\nBaktiar Laskar Y, Meitei Lourembam R, Behari Mazumder P:Herbal Remedies for Breast Cancer Prevention and Treatment. Medicinal Plants - Use in Prevention and Treatment of Diseases. IntechOpen;2020.\n\nChaiyana W, Anuchapreeda S, Punyoyai C, et al.: Ocimum Sanctum Linn. as a Natural Source of Skin Anti-Ageing Compounds. Ind. Crop. Prod. 2019; 127: 217â224. Publisher Full Text\n\nJambheshwar G: Theraupatic Potential and Phytopharmacology of Tulsi Psychopharmacological Studies to Identify Plants Possessing Anti-Anxiety Potential and to Develop a New Laboratory Model. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen D, Zhao J, Cong W: Chinese Herbal Medicines Facilitate the Control of Chemotherapy-Induced Side Effects in Colorectal Cancer: Progress and Perspective. Front. Pharmacol. 2018; 9: 9. Publisher Full Text\n\nShareef M, Ashraf MA, Sarfraz M: Natural Cures for Breast Cancer Treatment. Saudi Pharm. J. 2016; 24: 233â240. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUtispan K, Niyomtham N, Yingyongnarongkul B-E, et al.: Ethanolic Extract of Ocimum Sanctum Leaves Reduced Invasion and Matrix Metalloproteinase Activity of Head and Neck Cancer Cell Lines.Publisher Full Text\n\nHarsha M, Mohan Kumar K, Kagathur S, et al.: Effect of Ocimum Sanctum Extract on Leukemic Cell Lines:A Preliminary in-Vitro Study. J. Oral Maxillofac. Pathol. 2020; 24: 93â98. Publisher Full Text\n\nLuke AM, Patnaik R, Kuriadom ST, et al.: An in vitro Study of Ocimum Sanctum as a Chemotherapeutic Agent on Oral Cancer Cell-Line. Saudi J. Biol. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumar S, Kashyap P: Antiproliferative Activity and Nitric Oxide Production of a Methanolic Extract of <I>Fraxinus Micrantha</I> on Michigan Cancer Foundation-7 Mammalian Breast Carcinoma Cell Line. J. Intercult. Ethnopharmacol. 2015; 4: 109â113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMishra D, Patel V, Banerjee D: Nitric Oxide and S-Nitrosylation in Cancers: Emphasis on Breast Cancer. Breast Cancer (Auckl.). 2020; 14: 117822341988268. Publisher Full Text\n\nKustiati U, Wihadmadyatami H, Kusindarta DL: Dataset of Phytochemical and Secondary Metabolite Profiling of Holy Basil Leaf (Ocimum Sanctum Linn) Ethanolic Extract Using Spectrophotometry, Thin Layer Chromatography, Fourier Transform Infrared Spectroscopy, and Nuclear Magnetic Resonance. Data Brief. 2022; 40: 107774. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKustiati U, Dewi Ratih TS, Dwi Aris Agung N, et al.: In Silico Molecular Docking and in vitro Analysis of Ethanolic Extract Ocimum Sanctum Linn.: Inhibitory and Apoptotic Effects against Non-Small Cell Lung Cancer. Vet. World. 2021; 14: 3175â3187. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSundaram MK, Khan MA, Alalami U, et al.: Phytochemicals induce apoptosis by modulation of nitric oxide signaling pathway in cervical cancer cells. Eur. Rev. Med. Pharmacol. Sci. 2020; 24(22): 11827â11844. PubMed Abstract | Publisher Full Text\n\nKustiati U, Wihadmadyatami H, Kusindarta DL: Dataset of Phytochemical and Secondary Metabolite Profiling of Holy Basil Leaf (Ocimum Sanctum Linn) Ethanolic Extract Using Spectrophotometry, Thin Layer Chromatography, Fourier Transform Infrared Spectroscopy, and Nuclear Magnetic Resonance. Data Brief. 2022; 40: 107774. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAborehab NM, Elnagar MR, Waly NE: Gallic Acid Potentiates the Apoptotic Effect of Paclitaxel and Carboplatin via Overexpression of Bax and P53 on the MCF-7 Human Breast Cancer Cell Line. J. Biochem. Mol. Toxicol. 2021; 35: e22638. PubMed Abstract | Publisher Full Text\n\nAborehab NM, Osama N: Effect of Gallic Acid in Potentiating Chemotherapeutic Effect of Paclitaxel in HeLa Cervical Cancer Cells. Cancer Cell Int. 2019; 19: 154. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen CY, Kao CL, Liu CM: The Cancer Prevention, Anti-Inflammatory and Anti-Oxidation of Bioactive Phytochemicals Targeting the TLR4 Signaling Pathway. Int. J. Mol. Sci. 2018; 19. Publisher Full Text\n\nLeón-González AJ, Auger C, Schini-Kerth VB: Pro-Oxidant Activity of Polyphenols and Its Implication on Cancer Chemoprevention and Chemotherapy. Biochem. Pharmacol. 2015; 98: 371â380. PubMed Abstract | Publisher Full Text\n\nFuster MG, Carissimi G, Montalbán MG, et al.: Antitumor Activity of Rosmarinic Acid-Loaded Silk Fibroin Nanoparticles on Hela and MCF-7 Cells. Polymers (Basel). 2021; 13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNadeem M, Imran M, Gondal TA, et al.: Therapeutic Potential of Rosmarinic Acid: A Comprehensive Review. Appl. Sci. (Switzerland). 2019; 9: 9. Publisher Full Text\n\nAnwar S, Shamsi A, Shahbaaz M, et al.: Rosmarinic Acid Exhibits Anticancer Effects via MARK4 Inhibition. Sci. Rep. 2020; 10: 10300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJaved Z, Sadia H, Iqbal MJ, et al.: Apigenin Role as Cell-Signaling Pathways Modulator: Implications in Cancer Prevention and Treatment. Cancer Cell Int. 2021; 21: 21. Publisher Full Text\n\nSeo HS, Jo JK, Ku JM, et al.: Induction of Caspase-Dependent Extrinsic Apoptosis by Apigenin through Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) Signalling in HER2-Overexpressing BT-474 Breast Cancer Cells. Biosci. Rep. 2015; 35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu J, Cao XC, Xiao Q, et al.: Apigenin Inhibits HeLa Sphere-Forming Cells through Inactivation of Casein Kinase 2α. Mol. Med. Rep. 2015; 11: 665â669. PubMed Abstract | Publisher Full Text\n\nShendge AK, Chaudhuri D, Basu T, et al.: A Natural Flavonoid, Apigenin Isolated from Clerodendrum Viscosum Leaves, Induces G2/M Phase Cell Cycle Arrest and Apoptosis in MCF-7 Cells through the Regulation of P53 and Caspase-Cascade Pathway. Clin. Transl. Oncol. 2021; 23: 718â730. PubMed Abstract | Publisher Full Text\n\nPham TH, le Page Y , Percevault F, et al.: Apigenin, a Partial Antagonist of the Estrogen Receptor (Er), Inhibits Er-Positive Breast Cancer Cell Proliferation through Akt/Foxm1 Signaling. Int. J. Mol. Sci. 2021; 22: 1â17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi P, Zhou L, Zhao T, et al.: Caspase-9: Structure, Mechanisms and Clinical Application. Oncotarget. 2017; 8: 23996â24008. Publisher Full Text\n\nBoice A, Bouchier-Hayes L: Targeting Apoptotic Caspases in Cancer. Biochim. Biophys. Acta, Mol. Cell Res. 2020; 1867: 118688. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNational Center for Biotechnology Information:PubChem Compound Summary for CID 5280443. [Dataset]. Apigenin. 2023. Reference Source\n\nNational Center for Biotechnology Information:PubChem Compound Summary for CID 689043. [Dataset]. Caffeic Acid. 2023.Reference Source\n\nNational Center for Biotechnology Information:PubChem Compound Summary for CID 370. [Dataset]. Gallic Acid. 2023.Reference Source\n\nNational Center for Biotechnology Information:PubChem Compound Summary for CID 5281792. [Dataset]. Rosmarinic Acid. 2023.Reference Source\n\nRenatus M, Stennicke HR, Scott FL, et al.:Structure of cleaved, CARD domain deleted Caspase-9. [Dataset].2011.Reference Source\n\nErlanson DA, Lam J, Wiesmann C, et al.:Caspase-3 tethered to irreversible inhibitor. [Dataset].2011.Reference Source"
}
|
[
{
"id": "163541",
"date": "06 Mar 2023",
"name": "Muhammad Lokman MD Isa",
"expertise": [
"Reviewer Expertise Molecular Genetics & Human Reproductive Hormones Study",
"Pharmaceutical Industry",
"Biotechnology",
"Medical Pharmacology & Basic Pharmacology (Pharmacokinetic and Pharmacodynamic)"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments/suggestions:\nDMSO function is to stop the formation of formazan crystals? Supposedly the DMSO was inserted to solubilize the formazan. (Paragraph explaining cytotoxicity assay).\n\n\"This study did not use material from animals or humans as test samples, thus ethical approval was not required.\" Does the cell line use plant-based? I am suggesting the authors paraphrase the sentence. For example, ethical approval is not required since the cell lines used in this study are not involving primary cell lines. Or, just remove the ethical considerations from the manuscript. Or, just mention \"Not Applicable\" in the section.\n\nI am suggesting the authors add a conclusion or extract from the discussion into another section (if possible). If not, please add more input to conclude the manuscript. For instance, \" In conclusion, the ethanolic extract Ocimum sanctum Linn (EEOS). is a potential natural product that can be used to treat cancer. This study demonstrates EEOS capable to reduce the viability and adhesion of MCF-7 and T47D breast cancer cell lines. In addition, the morphology of the cancer cell lines is showing more holes or pits on the surface which indicates apoptosis is occurring. Moreover, when the cancer cell lines were treated with EEOS, the nitric oxide (NO) level is also increased. Furthermore, it has been found that several active sites of caspase bound potentially could lead to apoptosis. However, further studies to know more deeply the mechanisms of how ethanolic extract Ocimum sanctum Linn. exerts its anticancer activity are required.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9432",
"date": "03 Apr 2023",
"name": "Hevi Wihadmadyatami",
"role": "Author Response",
"response": "1. DMSO function is to stop the formation of formazan crystals? Supposedly the DMSO was inserted to solubilize the formazan. (Paragraph explaining cytotoxicity assay). Answer:  thank you very much for the reviewer's valuable correction and as per the reviewer's correction we already revise the sentence into ââŠ.then given Dimethyl sulfoxide (Sigma-Aldrich, Munich, Germany) 100 ÎŒl/well to dissolved the formation of formazan crystals during the experimentâŠ.â on the revised version of our manuscript. 2. This study did not use material from animals or humans as test samples, thus ethical approval was not required.\" Does the cell line use plant-based? I am suggesting the authors paraphrase the sentence. For example, ethical approval is not required since the cell lines used in this study are not involving primary cell lines. Or, just remove the ethical considerations from the manuscript. Or, just mention \"Not Applicable\" in the section. Answer:  thank you very much for the reviewer's valuable correction and as per the reviewer's correction we already revise the declaration into ââŠ.Ethical approval is not required since the cell lines used in this study are not involving primary cell lines.\" on the revised version of our manuscript. 3. I am suggesting the authors add a conclusion or extract from the discussion into another section (if possible). If not, please add more input to conclude the manuscript. For instance, \" In conclusion, the ethanolic extract Ocimum sanctum Linn (EEOS). is a potential natural product that can be used to treat cancer. This study demonstrates EEOS capable to reduce the viability and adhesion of MCF-7 and T47D breast cancer cell lines. In addition, the morphology of the cancer cell lines is showing more holes or pits on the surface which indicates apoptosis is occurring. Moreover, when the cancer cell lines were treated with EEOS, the nitric oxide (NO) level is also increased. Furthermore, it has been found that several active sites of caspase bound potentially could lead to apoptosis. However, further studies to know more deeply the mechanisms of how ethanolic extract Ocimum sanctum Linn. exerts its anticancer activity are required.\" Answer:  thank you very much for the reviewer's valuable correction and as per the reviewer's correction we already add the paragraph and add the additional conclusion in the revised version of our revision manuscript as: âIn conclusion, the ethanolic extract Ocimum sanctum Linn (EEOS). is a potential natural product that can be used to treat cancer. This study demonstrates EEOS capable to reduce the viability and adhesion of MCF-7 and T47D breast cancer cell lines. In addition, the morphology of the cancer cell lines is showing more holes or pits on the surface which indicates apoptosis is occurring. Moreover, when the cancer cell lines were treated with EEOS, the nitric oxide (NO) level is also increased. Furthermore, it has been found that several active sites of caspase bound potentially could lead to apoptosis. However, further studies to know more deeply the mechanisms of how ethanolic extract Ocimum sanctum Linn. exerts its anticancer activity are required.\""
}
]
}
] | 1
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https://f1000research.com/articles/12-136
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https://f1000research.com/articles/12-330/v1
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24 Mar 23
|
{
"type": "Genome Note",
"title": "The first genome sequence of Anopheles squamous from Macha, Zambia",
"authors": [
"Valerie T. Nguyen",
"Travis C. Collier",
"Sangwoo Seok",
"Xiaodi Wang",
"Monicah M. Mburu",
"Limonty Simubali",
"Mary E. Gebhardt",
"Douglas E. Norris",
"Yoosook Lee",
"Valerie T. Nguyen",
"Travis C. Collier",
"Sangwoo Seok",
"Xiaodi Wang",
"Monicah M. Mburu",
"Limonty Simubali",
"Mary E. Gebhardt",
"Douglas E. Norris"
],
"abstract": "Despite efforts to minimize the impacts of malaria and reduce the number of primary vectors, malaria has yet to be eliminated in Zambia. Understudied vector species may perpetuate malaria transmission in pre-elimination settings. Anopheles squamosus is one of the most abundantly caught mosquito species in southern Zambia and has previously been found with Plasmodium falciparum sporozoites, a causal agent of human malaria. This species may be a critical vector of malaria transmission, however, there is a lack of genetic information available for An. squamosus. We report the first genome data and the first complete mitogenome (Mt) sequence of An. squamosus. The sequence was extracted from one individual mosquito from the Chidakwa area in Macha, Zambia. The raw reads were obtained using Illumina Novaseq 6000 and assembled through NOVOplasty alignment with related species. The length of the An. squamosus Mt was 15,351 bp, with 77.9 % AT content. The closest match to the whole mitochondrial genome in the phylogenetic tree is the African malaria mosquito, Anopheles gambiae. Its genome data is available through National Center for Biotechnology Information (NCBI) Sequencing Reads Archive (SRA) with accession number SRR22114392. The mitochondrial genome was deposited in NCBI GenBank with the accession number OP776919. The ITS2 containing contig sequence was deposited in GenBank with the accession number OQ241725. Mitogenome annotation and a phylogenetic tree with related Anopheles mosquito species are provided.",
"keywords": [
"Anopheles squamosus",
"understudied malaria vector",
"Africa",
"Zambia"
],
"content": "Introduction\n\nAnopheles squamosus (Theobald, 1901; Figure 1) can be found across Africa1 and is of particular relevance to public health due to its implication in the spread of residual malaria cases. Anopheles squamosus is one of the most abundantly caught anopheline species in malaria vector surveillance studies in southern Zambia. However, it is understudied species because of its exophilic and zoophilic behaviours.2,3 Though they are predominantly associated as a zoophilic species, they have been discovered to have high anthropophily in southern Zambia.4 Additionally, there has been the detection of Plasmodium falciparum sporozoite and DNA, a causal agent of human malaria, in An. squamosus.2,5\n\nA and B have been reproduced with permission from Dr. Rebekah Kading (Colorado State University).21 C has been reproduced with permission from Walter Reed Biosystematics Unit (WRBU).22\n\nUnfortunately, there are two key barriers to pursuing the rigorous investigation of the epidemiologically important traits of this vector, such as host choice, biting behaviours, and dispersal capacity. First, An. squamosus is morphologically indistinguishable from An. cydippis at the adult stage. Although they are morphologically distinct as larvae, larvae are often difficult to locate in abundance. There are numerous examples of sympatric Anopheles sibling species expressing drastic differences in insecticide resistance6 or host choice.7,8 These differences make species confirmation critical to assessing and mitigating malaria transmission risk. Second, there is limited genetic information (173 sequences total in GenBank as of August 2022) for An. squamosus, most (N=166; 96%), are partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. ITS2 sequences are better at differentiating species within a complex than COI sequences9 but existing ITS2 primers do not typically work on An. squamosus and the absence of sequence data for this region prohibit the design of functional diagnostic PCR primers.\n\nTo overcome these two barriers and advance investigative efforts aimed at this widespread, yet neglected malaria vector, we carried out the first Illumina high-throughput sequencing of this species.\n\n\nMethods\n\nThe An. squamosus sample used for the genome sequencing was collected in Chidakwa near Macha, Zambia (utm-x: 0478202, utm-y: 8184394) using a CDC light trap placed outdoors near a goat pen. Samples were frozen after collection at â20°C until DNA extraction. DNA was extracted using a magnetic bead-based protocol optimized for mosquito DNA for Next-generation sequencing.10 The head and thorax were dissected from the sample and hydrated in nuclease-free water for 1 hour at 4°C. Tissues were then removed from the water and homogenised in a mixture of 2 ÎŒL Proteinase K (100 mg/mL) and 98 ÎŒL PK Buffer in a 1.5 mL Eppendorf microcentrifuge tube (add company name), followed by incubation at 56°C for 2 hours. The lysate was transferred to a new 1.5 mL microcentrifuge tube and mixed with a MagAttract Mix consisting of 100 ÎŒL isopropanol, 100 ÎŒL Buffer AL, and 15 ÎŒL MagAttract Suspension G (Qiagen, Hilden, Germany). The mixture was incubated at room temperature for 10 minutes and occasionally vortexed to ensure that the magnetic beads were evenly dispersed. The microcentrifuge tube containing the lysate was then moved to a magnetic bead separator until the liquid appeared clear. After a series of ethanol washes of magnetic beads, DNA was eluted from the beads with 100 ÎŒL AE Buffer and stored at â20°C until library preparation. The library preparation was completed using the QIAseq FX UDI kit (Qiagen, Hilden, Germany) using 20 ng genomic DNA as input for the protocol as previously described.11\n\nRaw sequencing reads were trimmed using fastp (RRID:SCR_016962) version 0.20.1.12 Mitogenome (Mt) contig was assembled using NOVOPlasty (RRID:SCR_017335) version 4.3.1.13 Automatic annotation of mitogenome was conducted with the MITOS website14 using the invertebrate genetic code for mitochondria under default settings. Some automatic annotations were not consistent with typical Anopheles mitochondrial gene start and/or end positions. Manual adjustments were made to inconsistent automatic annotations by shifting the start and end positions to match existing Anopheles mitochondrial gene annotations found in GenBank. Annotation information was also deposited to the GenBank with the genome sequence. The full genomic map is provided in Figure 2.\n\nPhylogenetic analysis was conducted using the mitogenome sequences of seven Anopheles species and one Aedes species as an outgroup in. The Jukes-Cantor model was used to calculate the pairwise genetic distances and the neighbour-joining method was used to build the phylogenetic tree in Geneious Prime (RRID:SCR_010519) 2022.02 (Biomatters, Auckland, New Zealand)15 (free alternative, AliView).\n\nDraft genome assembly was conducted using MaSuRCA (RRID:SCR_010691) version 4.0.916 in order to find a contig containing Internal transcribed spacer 2 (ITS2) sequence. Basic local alignment search tool (BLAST) (RRID:SCR_004870) was used for the resulting contigs to locate contigs with highest similarity with only An. squamosus ITS2 sequence available on GenBank (accession number MK592071).\n\n\nResults\n\nWe yielded 105 million reads from sequencing a single An. squamosus sample. Of these, 238,740 reads were used to assemble mitochondrial genome. Draft genome assembly using MaSuRCA produced 58,252 scaffolds with the total size of scaffolds of 350Mbp. N50 scaffold length was 21,439bp. Among these contigs, we identified one contig containing ITS2 region (GenBank accession number OQ241725), which was 1,223 bp long.\n\nThe length of the An. squamosus Mt (GenBank accession number OP77691923) was 15,351 bp and the percentage A+T was 77.9% (Figure 2). The average A+T percentage of eight other anopheline species was 77.7% (±0.61 SD). The length of this mitochondrial genome was a similar length to other anopheline species that have been deposited in GenBank, with the average of the eight species compared in this analysis being 15,363 bp. The content for this mitochondrial genome includes two ribosomal RNAs, 22 transfer RNAs, and 35 protein-coding genes. The cytochrome c oxidase I (COI) fragment spanning 1,462-2,132 bp of An. squamosus sequence had 97.7% (±4.27 SD, N=9) similarity to the COI sequence of An. squamosus deposited in GenBank.\n\nIn the phylogenetic analysis (Figure 3), the closest match to the whole mitogenome sequence of An. squamosus was the African malaria mosquito An. gambiae (GenBank accession number L20934), with 91.5% sequence similarity. This comparable sample was identified as An. gambiae and published in 1993 before An. gambiae were separated into two species: An. gambiae and An. coluzzii.17 Nevertheless, previous studies suggest that mitogenome sequence alone is not sufficient to distinguish An. gambiae s.s. from An. coluzzii.18,19\n\nSpecies names are provided next to the GenBank accession numbers. Numbers at nodes indicate bootstrap values out of 100 replicates. Aedes aegypti was considered as an outgroup. The scale bar indicates relative nucleotide difference (0.02=2% nucleotide difference).\n\nThis study provides a critical genomic resource for research of this understudied malaria vector. Our short reads sequencing data was not sufficient to assemble high-quality reference genome and revealed the need for alternative long-read sequencing technology for a high-quality genome assembly. However, we provided a key ITS2 region data that researchers can develop a low-cost molecular diagnostic assay to identify species. Currently available ITS2 primers for anopheline species identification typically does not produce a PCR amplicon, which is one of the major roadblocks in carrying out surveillance and research of this species. We identified the ITS2-containing contig (GenBank Accession number OQ241725) that could be used for new primer design that would amply the ITS2 fragments more reliably for An. squamosus. Our genome sequence data could be used for further variant identification once high-quality reference genome become available for An. squamosus. The mitogenome sequence could also be used to identify phylogenetic relationship within and between related species and infer gene flow/dispersal.9,20\n\nThe study involves collection of mosquito specimen near goat pens within individual households in Chidakwa, Zambia as part of the project that had been approved by National Health Research Authority, Zambia: Approval No: NHRA00016/18/08/2021.",
"appendix": "Data availability\n\nGenBank: Anopheles squamosus mitochondrion, complete genome. Accession number OP776919; https://identifiers.org/ncbi/insdc:OP776919. 23\n\nBioProject: Complete mitogenome sequence of Anopheles squamosus from Macha, Zambia. Accession number PRJNA896235; https://identifiers.org/bioproject:PRJNA896235. 24\n\nSRA: DNA-Seq of mosquito Anopheles squamosus. Accession number SRR22114392; https://identifiers.org/insdc.sra:SRR22114392. 25\n\nBioSample: Anopheles squamosus isolate As22MACHA01. Accession number SAMN31538381; https://identifiers.org/biosample:SAMN31538381. 26\n\n\nAcknowledgements\n\nWe thank UF ICBR for providing sequencing services. We appreciate the support from Dr. Edgar Simulundu from Macha Research Trust toward our project.\n\n\nReferences\n\nTheobald FV: A monograph of the Culicidae, or mosquitoes. London: British museum (Nat. hist.).Department of Zoology; 1901; 256.\n\nStevenson JC, Simubali L, Mbambara S, et al.: Detection of Plasmodium falciparum Infection in Anopheles squamosus (Diptera: Culicidae) in an Area Targeted for Malaria Elimination, Southern Zambia. J. Med. Entomol. 2016 Nov; 53(6): 1482â1487. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoffman JE, Ciubotariu SL, Mudenda T, et al.: Phylogenetic Complexity of Morphologically Identified Anopheles squamosus in Southern Zambia. Insects. 2021 Feb 8; 12(2). PubMed Abstract | Publisher Full Text | Free Full Text\n\nFornadel CM, Norris LC, Franco V, et al.: Unexpected anthropophily in the potential secondary malaria vectors Anopheles coustani s.l. and Anopheles squamosus in Macha, Zambia. Vector Borne Zoonotic Dis. 2011 Aug; 11(8): 1173â1179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStevenson JC, Norris DE: Implicating cryptic and novel anophelines as malaria vectors in Africa. Insects. 2017; 8(1): 1â18.\n\nMain BJ, Lee Y, Collier TC, et al.: Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali. Mol. Ecol. 2015 Oct; 24(20): 5145â5157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKent RJ, Thuma PE, Mharakurwa S, et al.: Seasonality, blood feeding behavior, and transmission of Plasmodium falciparum by Anopheles arabiensis after an extended drought in southern Zambia. Am. J. Trop. Med. Hyg. 2007 Feb; 76(2): 267â274. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMain BJ, Lee Y, Ferguson HM, et al.: The genetic basis of host preference and resting behavior in the major African malaria vector, Anopheles arabiensis. PLoS Genet. 2016 Sep; 12(9): e1006303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJones CM, Ciubotariu II, Muleba M, et al.: Multiple novel clades of anopheline mosquitoes caught outdoors in northern Zambia. Frontiers Trop. Dis. 2021; 2: 780664. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen T, Vorsino AE, Kosinski KJ, et al.: A magnetic-bead-based mosquito DNA extraction protocol for Next-Generation sequencing. J. Vis. Exp. 2021; 170: e62354. Publisher Full Text\n\nKelly ET, Mack LK, Campos M, et al.: Evidence of local extinction and reintroduction of Aedes aegypti in Exeter, California. Front. Trop. Dis. 2021 Jul; 2(8). Publisher Full Text\n\nChen S, Zhou Y, Chen Y, et al.: fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018 Sep 1; 34(17): i884âi890. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDierckxsens N, Mardulyn P, Smits G: NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 2017 Feb 28; 45(4): e18. PubMed Abstract | Publisher Full Text\n\nBernt M, Donath A, Juhling F, et al.: MITOS: improved de novo metazoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 2013 Nov; 69(2): 313â319. PubMed Abstract | Publisher Full Text\n\nKearse M, Moir R, Wilson A, et al.: Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012 Jun 15; 28(12): 1647â1649. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimin AV, Marçais G, Puiu D, et al.: The MaSuRCA genome assembler. Bioinformatics. 2013 Nov 1; 29(21): 2669â2677. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoetzee M, Hunt RH, Wilkerson RC, et al.: Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. Zootaxa. 2013; 3619(2): 246â274. Publisher Full Text\n\nHanemaaijer MJ, Houston PD, Collier TC, et al.: Mitochondrial genomes of Anopheles arabiensis, An. gambiae and An. coluzzii show no clear species division. F1000Res. 2018; 7: 347. Publisher Full Text\n\nFontaine MC, Pease JB, Steele A, et al.: Mosquito genomics. Extensive introgression in a malaria vector species complex revealed by phylogenomics. Science. 2015 Jan 2; 347(6217): 1258524. Publisher Full Text\n\nCampos M, Patel N, Marshall C, et al.: Population genetics of Anopheles pretoriensis in Grande Comore Island. Insects. 2023; 14(1): 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKent RJ: The Mosquitoes of Macha, Zambia. The third International Malaria Research Conference. Baltimore, MD, USA: 2006 [cited 2022 Nov 8]. Reference Source\n\nWalter Reed Biosystematics Unit (WRBU): Anopheles squamosus Theobald.1901. [cited 2022 Nov 3]. Reference Source\n\nNguyen VT, Gebhardt ME, Mburu MM, et al.: Anopheles squamosus mitochondrion, complete genome. [Dataset]. GenBank. 2023. Reference Source\n\nUniversity of Florida: Complete mitogenome sequence of Anopheles squamosus from Macha, Zambia. [Dataset]. BioProject. 2022. Reference Source\n\nUniversity of Florida: DNA-Seq of mosquito Anopheles squamosus. [Dataset]. SRA. 2022. Reference Source\n\nUniversity of Florida: Anopheles squamosus isolate As22MACHA01. [Dataset]. BioSample. 2022. Reference Source"
}
|
[
{
"id": "167805",
"date": "05 Apr 2023",
"name": "Megan A. Riddin",
"expertise": [
"Reviewer Expertise Medical entomology",
"vector control",
"malaria",
"vector-borne disease"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research presented the first genome data and complete mitogenome sequence of Anopheles squamosus. Anopheles squamosus is commonly caught in abundance in Zambia and has been recently implicated in malaria transmission through the detection of P. falciparum circumsporozoites and through PCR. This research utilised a single An. squamosus specimen from Chidakwa, Zambia, which was collected through a CDC light trap placed outside near a goat pen. DNA was extracted from the specimen following a magnetic bead-based protocol specific for mosquito DNA for next generation sequencing. The library was then prepared using the eluted DNA and a standardised preparation kit (QIAseq FX UDI Kit). The sequencing reads were trimmed and mitogenome assembled using NOVOPlasty, and automatic annotation completed through the MITOS website. Phylogenetic analyses were performed with an Aedes species outgroup in Geneious Prime. Final draft genome assembly was conducted using MaSuRCA. The methods resulted in 105 million reads and one contig was identified as containing an ITS2 region. The mitochondrial genome was assembled and resulted in 15351 bp with 77% A+T percentage. Phylogenetic analyses identified the closest mitogenome sequence match to An. gambiae which was sequenced prior to separation of the complex into sibling species.\n\nThe research enabled identification of key ITS2 region data for development of low-cost molecular diagnostic assays, despite the short reads not being sufficient for high-quality reference genome assembly. The outputs further included the identification of the phylogenetic relationships to related species through assembled mitogenome sequence.\nThis study presented vital data on an understudied species that has recently been implicated in malaria transmission in Zambia. Malaria has experienced recent upsurges in Zambia and although the primary vectors are thought to be An. funestus, An. gambiae and An. arabiensis, the detection of higher abundance of An. squamosus and An. coustani, as well as their unexpected anthopophillic behaviour has lent support to their role in transmission. The recent detection of both P. falciparum circumsporozoite in An. squamosus has highlighted the necessity to study this species. The use of full genome sequencing is highly beneficial not only for phylogenetic analysis but further for development of rapid molecular identification techniques as was exhibited in this study. This study was able to provide data that will assist towards classification and identification of the species to better understand ecology, epidemiology and therefore role of this species in malaria transmission in Zambia.\n\nThe Introduction and rationale of the study is clearly explained and may benefit from some clarification on the chromosomal types or mention of possible clades that have been eluded to. However, as such work and suggestions are unpublished, it would be very interesting to see how the authors progress with this research towards understanding the phylogeny of the understudied species.\n\nThe Methods and Results are clear and concise and the diagrams are further well structured and complimentary to the text.\nVery few edits and/or additions are suggested and these are included below:\nIntroduction, Line 3: \"However, it is an understudied...\"\n\nIntroduction, Paragraph 2: It would be helpful to the reader to have understand if there is information on An. cydippis. Is this known as a vector and does it have anthropophilic or zoophilic behaviours?\n\nIntroduction, Paragraph 2: No mention is made of the unpublished claims of Green & Hunt that state there are 5 chromosomal forms of An. squamosus and also the publications that elude to multiple clades. This may be interesting to include to lend support to how understudied the species is.\n\nData collection, Line 6: \"...Eppendorf microcentrifuge tube (add company name)....\" should be addressed.\n\nResults, paragraph 4: This paragraph requires some grammar editing such as \"..that could be used for new primer design that would amply the ITS2 ....\" should be corrected to \"amplify\".\nOverall this is a very interesting and important article and is recommended for indexing.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
},
{
"id": "200578",
"date": "11 Oct 2023",
"name": "Tara Roth",
"expertise": [
"Reviewer Expertise I am a vector ecologist with a specialization in mosquitoes and ticks. I am currently working on a project addressing the distribution of sister Anopheline species An. hermsi and An. freeborni in California and thus am familiar with the regions (CO1 and ITS2) being discussed in this paper"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article details the production of the An. squamosus full genome which is intended to provide a useful reference for future researchers to investigate the role this species may play in the ecologic cycle of malaria in Zambia. The article stresses that mitogenome sequences (predominantly CO1) are not sufficient to differentiate closely related species which is a consistent issue across Culicidae. They also stress that primers for the alternative region, ITS2, do not amplify An. squamosus. The article is well written, however there are a few typos that should be addressed. Under Methods -> Data Collection, the line \"Eppendorf microcentrifuge tube (add company name)\" should be fixed. The final paragraph of the results section could be tightened up a bit - there are a few minor grammatical errors (eg: \"The mitogenome sequence could also be used to identify phylogenetic relationship within...\")\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-330
|
https://f1000research.com/articles/12-329/v1
|
23 Mar 23
|
{
"type": "Review",
"title": "Extracellular vesicles as novel drug delivery systems to target cancer and other diseases: Recent advancements and future perspectives",
"authors": [
"Divya Ramesh",
"Shankar Bakkannavar",
"Vinutha R Bhat",
"Krishna Sharan",
"Divya Ramesh",
"Vinutha R Bhat",
"Krishna Sharan"
],
"abstract": "Extracellular vesicles (EVs) are lipid-bound vesicles produced into the extracellular space by cells. Apoptotic bodies (ApoBD), microvesicles (MVs), and exosomes are examples of EVs, which act as essential regulators in cell-cell communication in both normal and diseased conditions. Natural cargo molecules such as miRNA, messenger RNA, and proteins are carried by EVs and transferred to nearby cells or distant cells through the process of circulation. Different signalling cascades are then influenced by these functionally active molecules. The information to be delivered to the target cells depends on the substances within the EVs that also includes synthesis method. EVs have attracted interest as potential delivery vehicles for therapies due to their features such as improved circulation stability, biocompatibility, reduced immunogenicity, and toxicity. Therefore, EVs are being regarded as potent carriers of therapeutics that can be used as a therapeutic agent for diseases like cancer. This review focuses on the exosome-mediated drug delivery to cancer cells and the advantages and challenges of using exosomes as a carrier molecule.",
"keywords": [
"EXTRACELLULAR VESICLES",
"Exosomes",
"Signalling cascade",
"Biocompatibility",
"Biogenesis",
"Cancer"
],
"content": "Introduction\n\nIn multicellular organisms, interaction among the cells is required to sustain cellular activities and tissue homeostasis. Direct contact between the cells, are used to facilitate intercellular communication.1 According to Wolf et al., 1967, microvesicles, formerly known as âplatelet dust,â were initially recognised as a subcellular substance derived from thrombocytes that was present in both blood serum and plasma. These extracellular vesicles (EVs) possess importance in communication between the cells. These functions are done by transporting membrane and cytosolic proteins, lipids, and RNA between cells.1 Exosomes are a type of extracellular vesicles with a diameter of 40â150 nm that are produced by all cells and exchanged between them.2 Other vesicles engaged in intercellular communication include MVs, ectosomes, shedding vesicles, and microparticles.3â5 Exosomes are produced from various cells when multivesicular bodies merge with the plasma membrane.6,7 Natural sources of exosomes are saliva, blood, semen, urine, plasma, cerebrospinal fluid, and breast milk.8,9 Exosomes are typically distinguished by their size, as well as the expression of exosome marker proteins, such as CD63, CD81, and CD9, etc.10â12 EVs have been studied for their capacity to deliver drug to treat conditions like cancer and other diseases such as liver ailments,13 nerve disorders,14 sepsis treatment,15 and wound healing.16\n\nThere are three types of EVs, namely Exosomes, microvesicles, and apoptotic bodies. These are differentiated and classified according to size and method of biogenesis17,18â21 as depicted in Figure 1. Exosomes are endocytic in origin formed by plasma membrane budding and blebbing.22 The density, content, and function of EVs are further distinguished.22,23 The details of various vesicle subtypes are as shown in Table 1.\n\nExosomes are specific type of EVs that resembles structures of cells.33 They are now recognised for playing significant roles in cellular communication through various methods. Cellâcell communication occurs via the delivery of peptides, metabolites, and nucleic acids to targeted cells.\n\nExosomes have certain biological properties like immune response,34 repairing tissues,35,36 stem cell maintenance,37 etc. Biological properties of exosomes such as immune response regulation is due to the secretion of the extracellular vesicles from the dendritic cells (DC). This process helps in the transfer of peptides like major histocompatibility complex class II (MHC II) to other DC. Membrane proteins are also transferred intracellularly during the interaction between CD4+ and CD8+ cells. Exosomes derived from DCs have been found to be distinct intercellular vectors. Hence DC-derived exosomes aid in the regulation and maintenance of the immune response. Exosomes also help in the process of tissue repair after an injury. The process of tissue repair has been observed in the exosomes that are derived from mesenchymal stem cells (MSC-Ex). Mesenchymal stem cells derived from the human umbilical cord (HucMSC-Ex) prevent apoptosis and accelerate the process of healing by promoting the cell proliferation. Re-epithelialization of the cells during wound healing is due for the activation of Wnt/β-catenin derived from the HucMSC-Ex. During this process there will be an increased expression of proteins such as collagen I, PCNA (proliferative Cell Nuclear Antigen) and CK 19 (Cytokeratin 19) respectively that promotes wound healing and tissue repair. HucMSC-Ex are found to cure renal tube injuries and has a function in escalating the cellâcell communication.38 Treatment of a wound with HucMSC-Ex reverses AKT (AK Strain Transforming) inhibition and promotes proliferation of the cells thus helping in the healing process. Cellâcell communication by the exosomes also helps in maintenance of pluripotency in neighbouring cells including the embryonic stem cells (ES Cells). ES cells derived from the membrane vesicles or exosomes tend to express proteins responsible for the self-renewal and maintenance of stem cells and its pluripotency. Maintenance of pluripotency will be done by enhanced expression of proteins such as Oct-4, Nanog and Rex 1 respectively. Overexpression of Wnt along with these proteins also enhances the pluripotency of cells thus helps in maintaining the same throughout the generations. These vesicles are involved in various clinical applications such as carriers used to transport cargo for therapeutic purposes.39 These vesicles are preferred due to their reduced immunogenicity and structure. The structure of exosomes includes the closed lipid bilayer thus protecting them from the degradation of cargo. This membrane composition helps these to cross the membranes of the brain including the bloodâbrain barrier easily and freely compared to other carrier substances.40,41 Even though all these advantages make the exosomes an excellent vehicle for cargo transport, the major disadvantage is difficulty in targeting specific organs by reducing the off-target distribution.41â43\n\nOff target distribution of the exosomes is a major challenge in using exosomes as a delivery vehicle. To override this problem the exosomes can be surface modified easily by using chemical modification and genetic engineering. In the process of genetic engineering, the gene sequence of the guiding polypeptide will be fused with membrane proteins of the exosomes. This method is best suited for the surface display of only the membrane proteins on the exosomes. A wide range of surface modification can be achieved through chemical modification strategies.44 This method facilitates display of wide range of both natural and artificial ligands on the exosomes by assembly of fats. Reactions through conjugation will modify the exosomes in a stable manner, but the efficiency of this reaction will be reduced due to the intricacy of the proteins on the surface of exosomes. Assembly of the lipids on the exosomal membranes will cause exosome toxicity. Compared to the method of chemical modification, genetic engineering is more favoured due to its high advantages. It is the most convenient method to impose changes on the exosomes. Genetic engineering is done by the fusion of peptides or ligands of interest on the transmembrane proteins of the exosomes followed by transfection of the donor cells with an engineered plasmid, which facilitates the release of modified exosomes to the target cells or tissues. The lysosome-associated membrane protein (LAMP) family that includes LAMP-2B is currently the major exosomal surface protein used. LAMP-2B family is localized to endosomes and lysosomes with a very small fraction expressed on the exosomal surface (only N terminus).45 LAMP-2B has been used in targeting various tissues, for example, the RVG (rabies virus glycoprotein) peptide of rabies virus fused to LAMP-2B was effectively delivered to acetylcholine receptors and hence delivered drugs to specific areas of the brain. This has been used in targeted therapy of Alzheimerâs disease and it also helps in neurogenesis.46 LAMP-2B was also used in another experiment to deliver exosomes loaded with doxorubicin to target breast cancer cells through intravenous injection. Besides LAMP-2B, the tetraspanin superfamily CD63/CD9/CD81 are also used in exosome engineering. All these protein families can be used in surface modification and targeting; hence these can be used to prevent off-targeting.\n\nExosomes carries different types of cargoes that includes proteins, amino acids, lipid and its derivatives and various metabolites.47 Shape of exosomes varies from spherical shape when it is in solution, but it changes to biconcave, or cup-shaped during the phases of processing.48 The major cargo proteins include the tetraspanin class of proteins such as CD9, CD63, CD53, etc., that helps in targeting,49 ESCRT (Endosomal Sorting Complexes Repaired for Transport) proteins required an endosomal sorting complex is essential for the translocation, certain other peptides such as integrins, heat shock proteins (HSP), etc.50 as shown in Figure 2. Other than the common protein cargoes cell-specific peptides such as Major Histocompatibility Complex (MHC) class I and MHC class II depend on type of donor cells.51\n\nExosomes also contain cluster of nucleic acids such as deoxyribonucleic acid, ribonucleic acid, non-coding RNA and micro RNAs (miRs) make up one of the abundant types of RNAs in the exosome.52 The exosomal membrane also consists of many lipid derivatives such as ceramide, and sphingomyelin that help to sort cargoes, transportation, secretion, etc., ribosomal RNA (rRNA), long non-coding RNA (lncRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA, and p-element-induced wimpy testis (piwi)-interacting RNA are some of the other exosomal RNA species that have an impact on biological processes, particularly in tumour development.39,53\n\nMicrovesicles (ectosomes or microparticles) are a type of EV that gets produced from the cell membranes.54 In eukaryotes and other multicellular organisms microvesicles are also found in body fluids and tissues along with the other extracellular vesicles. Size of the microvesicles ranges from 30 nm to 1000 nm.55 Microvesicles function similar to other extracellular vesicles in the process, such as tumour immune supression, metastasis, angiogenesis etc.56 Microvesicles also helps in the removal of proteins misfolded during protein translocation and it helps in the removal of cell wastes or cell debris from the cellular microenvironment.57 Cancer growth and metastasis can be determined from the level of microvesicles in the body. Hence microvesicles can also be used as biomarkers.\n\nLarge number of molecules are present inside the microvesicles. Microvesicles do not contain organelles like that of the cells. It is devoid of nucleus, golgi, endoplasmic reticulum etc. Hence microvesicles are not regarded as the intact cells.58,59 Membranes of the microvesicles are made of membrane lipids as well as proteins. Main function of all the microvesicles is membrane transport and fusion. The membrane is made of phopholipid bilayer and also houses different kinds of proteins.58\n\nSome proteins are specific to the origin while some are common to all the microvesicles. Some tetraspanin family of proteins such as CD9, CD37, and CD63 can be seen on the surface of microvesicles.58â61 These surface markers helps in the protein sorting and selection of specific cargoes into lumen of microvesicles or membrane.62 In addition to lipids ad peptides microvesicles also contains some nucleic acid such as messenger RNA (mRNA) and microRNA (miRNA).61\n\nCells undergoing the process of apoptosis release certain extracellular vesicles called as apoptotic bodies. Its size ranges from 500 nmâ2 ÎŒm and they are more in number compared to other extracellular vesicles. Appearance of apoptotic bodies is an indicator of cell death or apoptosis while the other extracellular vesicles such as exosomes and microvesicles plays a major role in intercellular trafficking.63 A single cell may produce large number of apoptotic bodies during the apoptosis. Apoptotic bodies (ApoBDs) varies in the content as well as in the size.64 The contents within the apoptotic bodies varies accordingly such as chromatin remnants, some parts of chromatin, some organelles or portions of it.65 Apoptotic bodies are one of the least studied extracellular vesicles. Apoptotic bodies forms during the process of apoptosis and large number of apoptotic bodies will be released during the death of a single cell. Apoptotic bodies are engulfed by macrophages or cancer cells after being released in extracellular space and are then broken down in phagolysosomes. âTingible body macrophagesâ are macrophages that consume apoptotic cells.66,67\n\nRemoval of apoptotic bodies doesnât produce any inflammatory reactions within the body as there is no release of any free contents or cellular debris to the extracellular environment. Once the ApoBDs are produced they will be engulfed by phagocytes thus preventing the process of necrosis. The phagocytes after the engulfment doesnât produce certain inflammatory cytokines thus preventing any complications in the body during the apoptosis and formation of ApoBDs.68,69\n\nCells like dendritic cells, stem cells, macrophages, etc. will secrete exosomes.70 Exosomes are widely distributed and found in various body fluids such as synovial fluid, breast milk, urine, blood plasma, saliva, and amniotic fluid.71 The biodistribution of exosomes is dependent on the mode of administration of the same.70 The administration through the intravenous route results in biodistribution of the exosomes to various organs such as the liver, kidney, lungs, and spleen,70 and this intravenous injection of exosomes will result in rapid clearance from the bloodstream, but other methods like intranasal allows the fast transport to the brain.40,72 Therefore, exosomes are believed to be used as one of the best vehicles for targeted drug delivery. The size of exosomes also matters when targeting specific locations as the larger exosomes will get trapped in lymph nodes, bones as well as in liver.73 Targeting the exosomes to a specific area is important for the delivery of exosome contents to the target. Targeted delivery is often dependent on the markers present on surface of exosomes.74\n\nAlthough circulating microparticles are seen within blood of the individuals without any disease conditions. The levels of microvesicles drastically increase during certain diseases like hypertension, cardiovascular diseases75 and pre-eclampsia76 etc. In some instances the surface molecules of the endothelial micoparticles change, thus it can be regarded as the indicator of various diseases. Microvesicles are shed into the outer environment by membrane fission by formation of invaginating bodies to the outer environment. Budding of these extracellular vesicles depends on the specific locations depending on the type of proteins and lipids incorporated. Microvesicles are released at the specific location after the cargo loading.77\n\nApoptotic bodies are a special types of extracellular vesicles that are released from the cell that is undergoing apoptosis. ApoBDs are specifically formed from the plasma membrane through the process of membrane blebbing. These structures are found throughout the body during the apoptosis and it is also a carrier of DNA, RNA, and lipids etc. and are considered as tiny sealed pouches.78\n\nOnce extracellular vesicles has been released from the donor cells or tissues, it will interact with the target tissues through endocytosis. Once the EVs has entered, it releases cargo into the target cells.79\n\nExosomes on reaching the target sites interacts with receptors on cell membrane or it will be internalized. The cell surface peptides bind with the surface ligands of EV and trigger a cascade of reactions to activate the target cells. This process is used in the process of immunomodulation and apoptosis respectively.80\n\nEV uptake can also be done through the mechanism of membrane fusion, where the fusion of two hydrophobic membranes occurs. During this process, EVs interact with cells through plasma membrane fusion and content release. Fusion of membranes are employed by SNARE (Soluble N-Ethylmaleimide Sensitive Factor Activating Protein receptor) and RAB (Ras- Associated Binding) proteins81,82 The various methods through which EVs are taken up by the cells is depicted in Figure 3.\n\nAnother method by which exosome components are delivered to target cells is through internalization process. Endocytic pathways are used in the process of internalization where the contents of the exosomes are delivered to target cells.83â85 The process of cellular uptake is temperature sensitive and is directly proportional to the temperature fluctuations.\n\nEndocytosis mediated by clathrin molecules is characterized by the involvement of receptors and ligands. This method of internalization occurs in various types of cells like ovarian and colon tumour cells,74,86,87 cardiomyocytes,88 hepatocytes,89 macrophages,89,90 etc. are intenalized by clathrin protein. Dynamin-2 protein plays an important role in clathrin-mediated endocytosis and helps in the scission of the vesicle by forming a collar shaped structure in the neck of invagination. Inhibition of this protein shows reduced exosome uptake by macrophages.90â92 In certain cancer cell types, exosome uptake is enhanced by a transferrin receptor protein. This exosome uptake pathway is mediated by exosome and cargo composition. Caveolin dependent endocytosis (CDE) also has an important function in internalization. In CDE, plasma membrane forms tiny, cave-like invaginations called caveolar vesicles that are eventually torn off and absorbed into the cell. The cell membrane contains caveolin that forms glycolipid rafts like cholesterol, sphingolipid. Caveolin proteins help in the formation of caveolae and for the generation of caveolin rich rafts caveolin has to be oligomerized.93 A particular type of caveolin protein called caveolin 1 is considered as important for the EV uptake94 and its inactivation resulted in defective uptake or no uptake.\n\nRuffled extensions of the plasma membrane during macropinocytosis or cell drinking causes the internilization of EVs thus releasing the cargo into specific locations. For the process of macropinocytosis to occur, it requires the activation of Rac1 GTPase for the invagination and also it requires proper functioning of Na+/H+ exchanger and cholesterol.95,96 It has been observed that impairment of the same results in the failure of EV uptake.\n\nPhagocytosis is also considered as one of the methods by which EV uptake occurs into the cells. Phagocytosis or âcell eatingâ is opposite to macropinocytosis or âcell drinkingâ. Unlike pinocytosis, this process does not require ruffles of the membrane for the engulfment of EVs, instead it involves the formation of membrane invaginations. Compared to other modes of EV uptake, phagocytosis is regarded as the most effective.97\n\nEVs secreted from various types of cells through different mechanisms has proved to be biologically important. Extracellular vesicles are most popularly known as the carrier vehicles for most of the cargoes such as nucleic acids, drugs,proteins etc; and it delivers these substances to the specific receptor cells. Hence these nanocarriers are regarded as the most effective vehicles for the transportation of biological agents. It has been observed that the function of EVs depend upon the type of cargoes they carry. EVs carrying certain RNAs or long non coding RNAs help in the development of certain types of cancers.98 Even though some studies proved that cargo carried by EVs results in cancer development, some studies have proved that these cargo loaded EVs help in treating cancer.99 Function of EVs also depends on the cell of origin. EVs produced from some cancer cells promotes cell communications, thus producing specific signals for apoptosis, cell migration, growth etc.100 Thus EVs aid in the determining the fate of certain cells. Function of EVs also depends upon the nature of the cargoes carried by them. In a study conducted by Xue et al., 2017, it was explained that EV loaded with circular ribonucleic acid plays a role in the proliferation of Hepatocellular carcinoma cells.101 It was observed that the expression of some biomarkers such as cyclin D1 and EZH2 (Enhancer of Zeste Homolog) were increased rapidly due to the presence of circRASSF2 in the case of laryngeal squamous carcinoma (LSCC). Thus it was confirmed that cargo loaded EVs has the capacity to control the growth of the cells.102 Role of EVs in promoting autophagy and apoptosis has been a milestone in the treatment of cancer. EV loaded with certain anticancerous substances has been proved to promote cell death in HCC as well as in Glioblastoma.103\n\nRole of extracellular vesicles in pathogenesis\n\nAccording to Zitvogel et al., 1998, EVs such as exosomes obtained from the Dendritic cells and can express major histocompatibility complex I. Exosomes are also involved in various disease conditions such as a variety of cardiovascular physiological and pathological conditions.104,105 Recent research has found exosomes released from mammalian cells with normal physiology contributed to cellâcell communication in cardiac tissues. These vesicles consist of proteins, lipids, and genetic materials. Vesicles released by the cardiac cells consist of APOBDs, MVs and exosomes.106 It has been observed that exosomes are released from different cells within the heart and hence they are involved in various cardiac pathophysiological conditions.\n\nIt was noted that EVs play a major role in development of atherosclerosis. Atherosclerosis mainly occurs due to endothelial dysfunction due to impediment in the normal blood flow through the endothelium. Endothelial dysfunction occurs due to the increased secretion of oxidized LDL and homocysteine in the serum. According to Tsuda, 2015, ox-LDL and Hcy release the HSP70-containing exosomes from cultured aortic ECs which in turn induces the activity of monocytes through the proinflammatory differentiation of macrophages, further resulting in the development of atherosclerosis.107 Once atherosclerosis has formed, the blood vessels will be narrowed due to subsequent accumulation of plaque over years. The plaques once formed lead to irregular blood flow, leading to blood clots, and finally complete occlusion of the artery, finally leading to myocardial ischaemic injury. Myocardial ischaemic injury is caused due to alterations in the circulation of micro-RNA and the majority of circulating miRs are found enclosed within the EVs. Under any conditions of stress, fibroblasts promote the pathological hypertrophy of cardiac cells through exosomes and their micro-RNA load. In peripartum cardiomyopathy (PPCM), the elevation of miR-146a in endothelial cells blocks the blood vessel formation and increased release of endothelial cells derived miR-146a-enriched exosomes. The EVs once released from the endothelial cardiomyocytes results in the advancement of levels of miR-146a. This process will cause reduced expression of miR-146a target proteins like Erbb4, Notch1, and Irak1 in cardiomyocytes that causes decreased metabolic function. From these findings, it can be inferred the miRNA-based cell interaction of endothelial cells and cardiac muscle cells involves EVs, and hence EVs are also responsible for various cardiac pathologies.\n\nEVs has a role in the pathogenesis of neurodegenerative disorders. These disorders include the deposition of a specific protein in neuroanatomical locations. It is proved that major proteins involved in the pathology of these diseases are transported by the exosomes. The cerebrospinal fluid and blood of the neurodegenerative diseases affected individuals contain exosomes loaded with aggregated proteins that are specific to these diseases. Moreover, exosomes provide a favourable condition for protein transport into the brain.108\n\nThe role of EVs in cancer involves the transfer of cancer-causing proteins and nucleic acids that mediates the activities of the cells like its role in tumourigenesis.109 EVs obtained from the tumour cells facilitate tumour angiogenesis by converting the fibroblast and mesenchymal cells into myofibroblast. EVs isolated from the tumours can move neutrophils and skew the M2 polarization of the macrophages that helps in promoting the metastasis of the tumours. These exosomes also help in the development of multidrug resistance to the tumour cells by transferring certain MDR proteins and miRNAs as well as exporting drugs against and by neutralizing antibody-based drugs.113 Extracellular vesicles can thus play a dual function in cancer regulation, preventing or boosting growth, based on their bioactive payload and cell from where it originated.\n\nSince it has been proved that EVs unload their contents into the target cells, it has developed attraction in the field of oncotherapy.110â112 Various methods are being used in recent times to cure cancer using extracellular vesicles as described below:\n\nUsing EVs derived from the immune cells to suppress the growth of cancer cells\n\nIn cancer immunity, the dendritic cells are involved in the inhibition of tumour growth and metastasis, and this is done by capturing the new antigens or foreign bodies and hence producing the marked antibody response that involves cytotoxicity to specific tumour.113 EVs derived from the dendritic cells contain specific cargoes that are responsible for the antigen presentation and hence it acts as a perfect treatment for cancer.114 Zitvogel et al., 1998,115 suggested that tumour peptide-pulsed dendritic cell-derived exosomes could activate the cytotoxic T cells-based activity to reduce the metastatic growth in mouse that occurs within the body. In a study done by Munich et al., 2012, it was observed that dendritic cells kill the tumour cells and activate the natural killer cells through the TNF family ligands.116\n\nInhibition of cancer derived EVs release\n\nEVs derived from the cancer cells cannot be used for the treatment of cancer as it will accelerate the growth of cancer by providing favourable conditions for metastasis, immune escape, angiogenesis, drug resistance, etc.89,90 Therefore, the effective treatment of cancer can be achieved by inhibiting the synthesis, release, and uptake of the cancer derived EVs.117 Another technique to slow tumour growth is to use a haemofiltration system to remove circulating EVs. Tumours secrete a large number of EVs such as exosomes into the extracellular space, and it carries oncoproteins, immune suppressive molecules that help in tumour progression and metastasis. Several strategies have been proposed for targeting the malignant activities of tumours. Haemofiltration of the EVs is done to remove the circulating EVs from the blood. It is done using an affinity plasmapheresis platform known as adaptive dialysis-like affinity platform technology (ADAPT TM) system that helps to override the toxicity risks interactions of the drugs. This technique uses certain affinity agents that includes lectins and antibodies binding to EVs. These agents will be immobilized in the outer capillary space of plasma filtration membranes. This setup is like existing kidney dialysis system. Working principle for this setup is, when the patientâs blood passes through this device, the plasma components that are <200 nm will move through the porous fibres through the capillary action to interact with the substances that are immobilized on the device. The target molecules will bind tightly to the agent while other components of the blood will pass through the membrane. This device is multifaceted since large number of antibodies and other high affinity substances such as ligands can be absorbed into the cartridges to capture single or multiple targets. Although ADAPTTM therapies necessitate vascular surgery for patients, this subtractive approach to treat cancer using exosomes would avoid drug toxicity or any interaction hazards, thus giving it an edge over other pharmacological methods. As a result, the concept of this device offers a method for targeting exosomes that needs to be investigated for its potential as an additional therapeutic candidate to current cancer therapy protocols. This alternative could also be used as a therapeutic option for curing immunological dysfunction and improving immune responses to tumour growth.118\n\nEVs as carriers of genes\n\nEVs are being regarded as the effective carriers of the gene, but the usage of exosomes derived from the natural sources are difficult to study and expected therapeutic effects are achieved very rarely. The EVs that are modified through genetic engineering are more effective against cancer. EVs can also be used as miRNA carriers for effective treatment against cancer.119 miRNAs are tiny noncoding RNAs, and these are small and endogenous in nature. These miRNAs are used as an effective tool for cancer therapy. The major drawback of these miRNA mediated delivery is that miRNAs molecules get destroyed quickly in in vivo conditions. Therefore, the delivery of miRNA to specific location is considered as a difficult task. But if these miRNAs are encapsulated within the EVs, these can be delivered at the target site.120â124 EVs enriched with the anti-glioma miRNA (miRNA-146b) can decrease growth of glioma in the laboratory conditions according to Katakowski et al., 2013.125 EVs enriched in miR-101 can also inhibit osteosarcoma cell invasion/migration in vitro and metastasis in vivo.46 EVs are also being used as protein carriers for cancer therapy, where these can be loaded with tumour antigens,126 proteins that induce apoptosis,127 mutant and deficient proteins in apoptosis, several nanobodies,128 transferrin, and lactoferrins,129 proteasomes129 etc. for targeted therapy. Dendritic cells are used for T cell mediated immunotherapy by presenting the antigen from tumour to naïve T cells. But due to the low life span of DCs its use is limited.130 When compared to antigen presenting DCs, the exosomes isolated from the dendritic cells consists of complexes made from major histocompatibility factors and peptides and some co-stimulatory molecules on their membrane, that allows them to continue with the antigen presentation and enhance the process of immunisation in murine models.131 Survivin, an anti-apoptotic protein, suppresses the activation of cell death in large number of cancer cells.\n\nAccording to the studies conducted by Aspe and wall in 2010, it was observed that Survivin-T34A has an inactive mutation that reduces its pro-survival function that causes caspase activation followed by death of tumour cells.132\n\nEVs as carriers of chemotherapeutic drugs\n\nAnti-tumour chemotherapeutic medicines can successfully kill tumour cells that are rapidly developing. However, these medications have the potential to harm normal, healthy, fast-growing cells, resulting in major side effects. Furthermore, it is difficult for some hydrophobic medicines to target tumour cells with specificity. As a result, these formulations need more specific and efficient carrier. EVs have the capacity to be an efficient carrier for chemotherapeutic drugs as a result of their stable lipid bilayer structure.119 Chemotherapeutic drug-loaded exosomes particularly targeting cancer stem cells in vivo are the best exosome cancer therapy. Exosome-mediated chemotherapeutic administration has been proven to have significantly better anti-tumour effects in animal tumour models than free medicines. Doxorubicin, for example, is a frequent chemotherapy medication to treat haematolog ical cancers and a variety of solid tumours.133 Paclitaxel is another antimitotic chemotherapeutic medication that is commonly used in cancer treatment.134,135 Sonication can be used to load paclitaxel into exosomes, and it has proved to be 50 times more cytotoxic than the free paclitaxel in drug-resistant cancer cells in vitro. In a rat model of Lewis lung carcinoma, they can also significantly reduce tumour size and stop pulmonary metastasis.136 This suggests that paclitaxel contained in exosomes can target drug-resistant CSCs directly. Furthermore, paclitaxel-loaded exosomes from prostate cancer cells had increased cytotoxicity in autologous cancer cells.137 Drug-pre-treated donor cells can also create drug-loaded exosomes, which is interesting. EVs produced from mesenchymal stem cells treated with paclitaxel, for example, had a significant suppressing activity on tumour cells of pancreas.138\n\nEven though extracellular vesicles contribute to the pathology of some diseases it is also used as a therapeutic agent against certain diseases other than cancer. EVs loaded with cargo have proved to be effective against some diseases. Upadhya and Shetty, 2021, observed the therapeutic effects of EVs on the Parkinsons disease model (PD). It was studied that MSC -derived EVs protect dopaminergic neurons thus reducing apoptosis. This leads to the improved motor function in the Parkinson disease models. Hence it has been proved that EVs are effective against neurodegenerative diseases.139\n\nEVs also contributes to the protection of cardiac cells thus acting as a cardioprotective agent. EVs obtained from stem cells provide cardio protection, thus acting as a therapeutic tool against cardiovascular diseases. Stem cell-derived preconditioned cardioprotective exosomes are believed to be involved in cardio protective mechanisms. Hence Exosomes play an important role in cardioprotective as well as pathological conditions of heart cells.105\n\nEVs are also known to provide protection against inflammatory bowel disease (IBD) produced from dextran sodium sulfate through intestinal stem cell and epithelial regeneration. It was observed that EVs derived from the MSC-derived EVs resulted in normal functioning by downregulating inflammatory responses, thus promoting the regeneration of epithelial cells or tissues. Hence EVs can be used as a medicine for treating IBD.140\n\nThere are lots of challenges in using EVs as a delivery vehicle. Firstly there are no effective methods for isolation of EVs from various sources. Nowadays EVs are isolated using chemicals such as polyethylene glycol (PEG) which provides low specificity but high recovery. EVs isolated using ultracentrifugation method, size-based isolation techniques; immunoaffinity capture-based techniques; precipitation; microfluidics-based isolation technique provides medium recovery and specificity. Immunoaffinity based techniques and isolation using microfluidics are considered as low recovery with high specificity. Hence there is a requirement of high yielding, more economical and time saving method for the isolation.141\n\nSecond challenge is limited drug loading efficiency of EVs compared to other nanoparticles like liposomes. The reduced efficiency causes difficulty in loading exogenous drug into the EVs thus making it a great barrier in using the same for other applications. This may be due to its small size and reduced capacity compared to liposomes or other nanovesicles. This can be resolved by using efficient drug loading strategies like transfection, co-incubation for longer time, electroporation, rapid freeze thaw cycles.141\n\nThe third major challenge is instability of EVs when stored for longer time. It has been observed that EVs that are stored for longer time in â80°C loses its viability and efficiency within 2 weeks. Hence, there is a requirement of more efficient storage method that will preserve EVs for longer duration without degrading it.142\n\n\nConclusion and future perspectives\n\nEVs, as a natural carrier, is developing as a promising agent in cancer treatment. EV based cancer therapies include employing naturally produced immune cell exosomes to reduce tumour cells, blocking cancer cell-derived EV activity, and exploiting EVs as cargo carriers. Using EVs as a potent carrier protein involves lots of advantages as well as challenges. The advantages are EVs are less toxic and immunogenic than synthesized nanoparticles because they are more biocompatible and biodegradable.143 Although other extracellular vesicles obtained from different cells are biologically compatible, they are bigger and more ubiquitously distributed than exosomes, which limits their use in loading and distribution of drug. EVs are very simple to produce because most cell types can manufacture them. Additionally, EVs are present in almost all the body fluids, and their small size allows them to bypass lung clearance and cross the bloodâbrain barrier.144,145 Even though there are lots of advantage of using exosomes as carriers, the challenges are the differentiation between exosomes from various sources is still unclear. The number of EVs required to provide a therapeutic impact may range dramatically amongst malignancies. Tumour scalability and heterogeneity may have an impact on treatment outcomes and many other roles of EVs produced from various sources have not been thoroughly investigated. Furthermore, nothing is known about the exosome modifications that help in providing a high degree of selectivity for specific cancer cells. The storage and stability of EVs are still unknown.\n\nEVs offer novel and important applications in treating cancer and other diseases, despite the fact that there are many more challenges in using exosomes as carrier agents.",
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Control. Release. 2015; 207: 18â30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMathieu M, Martin-Jaular L, Lavieu G, et al.: Specificities of Secretion and Uptake of Exosomes and Other Extracellular Vesicles for Cell-to-Cell Communication. Nat. Cell Biol. 2019; 21(1): 9â17. PubMed Abstract | Publisher Full Text\n\nBunggulawa EJ, Wang W, Yin T, et al.: Recent Advancements in the Use of Exosomes as Drug Delivery Systems 06 Biological Sciences 0601 Biochemistry and Cell Biology. J. Nanobiotechnology. 2018; 16(1): 1â13. Publisher Full Text\n\nHa D, Yang N, Nadithe V: Exosomes as Therapeutic Drug Carriers and Delivery Vehicles across Biological Membranes: Current Perspectives and Future Challenges. Acta Pharm. Sin. B. 2016; 6(4): 287â296. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiang Y, Duan L, Lu J, et al.: Engineering Exosomes for Targeted Drug Delivery. Theranostics. 2021; 11(7): 3183â3195. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTerasawa K, Tomabechi Y, Ikeda M, et al.: Lysosome-Associated Membrane Proteins-1 and -2 (LAMP-1 and LAMP-2) Assemble via Distinct Modes. Biochem. Biophys. Res. Commun. 2016; 479(3): 489â495. PubMed Abstract | Publisher Full Text\n\nYang J, Zhang X, Chen X, et al.: Exosome Mediated Delivery of MiR-124 Promotes Neurogenesis after Ischemia. Mol. Ther. Nucleic. Acids. 2017; 7: 278â287. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBobrie A, Colombo M, Raposo G, et al.: Exosome Secretion: Molecular Mechanisms and Roles in Immune Responses. Traffic. 2011; 12(12): 1659â1668. Publisher Full Text\n\nYellon DM, Davidson SM: Exosomes: Nanoparticles Involved in Cardioprotection?. Circ. Res. 2014; 114(2): 325â332. Publisher Full Text\n\nZhao L, Gu C, Gan Y, et al.: Exosome-Mediated SiRNA Delivery to Suppress Postoperative Breast Cancer Metastasis. J. Control. Release. 2020; 318: 1â15. PubMed Abstract | Publisher Full Text\n\nGarcia NA, Ontoria-Oviedo I, González-King H, et al.: Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells. PLoS One. 2015; 10(9): e0138849. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMashouri L, Yousefi H, Aref AR, et al.: Exosomes: Composition, Biogenesis, and Mechanisms in Cancer Metastasis and Drug Resistance. Mol. Cancer. 2019; 18(1): 1â14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang X, Yuan T, Tschannen M, et al.: Characterization of Human Plasma-Derived Exosomal RNAs by Deep Sequencing. BMC Genomics. 2013; 14(1): 1â14. Publisher Full Text\n\nGe L, Zhang N, Li D, et al.: Circulating Exosomal Small RNAs Are Promising Non- invasive Diagnostic Biomarkers for Gastric Cancer. J. Cell. Mol. Med. 2020; 24(24): 14502â14513. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYáñez-Mó M, Siljander PRM, Andreu Z, et al.: Biological Properties of Extracellular Vesicles and Their Physiological Functions. J. Extracell Vesicles. 2015; 4(2015): 1â60. Publisher Full Text\n\nvan der Pol E , Böing AN, Harrison P, et al.: Classification, Functions, and Clinical Relevance of Extracellular Vesicles. Pharmacol. Rev. 2012; 64(3): 676â705. PubMed Abstract | Publisher Full Text\n\nCastellana D, Zobairi F, Martinez MC, et al.: Membrane Microvesicles as Actors in the Establishment of a Favorable Prostatic Tumoral Niche: A Role for Activated Fibroblasts and CX3CL1-CX3CR1 Axis. Cancer Res. 2009; 69(3): 785â793. PubMed Abstract | Publisher Full Text\n\nDhondt B, Rousseau Q, de Wever O , et al.: Function of Extracellular Vesicle-Associated MiRNAs in Metastasis. Cell Tissue Res. 2016; 365(3): 621â641. PubMed Abstract | Publisher Full Text\n\nPap E, Pállinger Ã, Pásztói M, et al.: Highlights of a New Type of Intercellular Communication: Microvesicle-Based Information Transfer. Inflamm. Res. 2009; 58(1): 1â8. PubMed Abstract | Publisher Full Text\n\nSchorey JS, Bhatnagar S: Exosome Function: From Tumor Immunology to Pathogen Biology. Traffic. 2008; 9(6): 871â881. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimpson RJ, Jensen SS, Lim JWE: Proteomic Profiling of Exosomes: Current Perspectives. Proteomics. 2008; 8(19): 4083â4099. PubMed Abstract | Publisher Full Text\n\nValadi H, Ekström K, Bossios A, et al.: Exosome-Mediated Transfer of MRNAs and MicroRNAs Is a Novel Mechanism of Genetic Exchange between Cells. Nat. Cell Biol. 2007; 9(6): 654â659. PubMed Abstract | Publisher Full Text\n\nRaposo G, Stoorvogel W: Extracellular Vesicles: Exosomes, Microvesicles, and Friends. J. Cell Biol. 2013; 200(4): 373â383. 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PubMed Abstract | Publisher Full Text\n\nNagata S, Suzuki J, Segawa K, et al.: Exposure of Phosphatidylserine on the Cell Surface. Cell Death Differ. 2016; 23(6): 952â961. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoon IKH, Lucas CD, Rossi AG, et al.: Apoptotic Cell Clearance: Basic Biology and Therapeutic Potential. Nat. Rev. Immunol. 2014; 14(3): 166â180. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKalluri R, LeBleu VS: The Biology, Function, and Biomedical Applications of Exosomes. Science. 2020; 367(6478). PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhite IJ, Bailey LM, Aghakhani MR, et al.: EGF Stimulates Annexin 1-Dependent Inward Vesiculation in a Multivesicular Endosome Subpopulation. EMBO J. 2006; 25(1): 1â12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhuang X, Xiang X, Grizzle W, et al.: Treatment of Brain Inflammatory Diseases by Delivering Exosome Encapsulated Anti- Inflammatory Drugs from the Nasal Region to the Brain. Mol. Ther. 2011; 19(10): 1769â1779. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang H, Freitas D, Kim HS, et al.: Identification of Distinct Nanoparticles and Subsets of Extracellular Vesicles by Asymmetric Flow Field-Flow Fractionation. Nat. Cell Biol. 2018; 20(3): 332â343. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoribe S, Tanahashi T, Kawauchi S, et al.: Mechanism of Recipient Cell-Dependent Differences in Exosome Uptake. BMC Cancer. 2018; 18(1): 1â9. Publisher Full Text\n\nBoulanger CM: Microparticles, Vascular Function and Hypertension. Curr. Opin. Nephrol. Hypertens. 2010; 19(2): 177â180. Publisher Full Text\n\nLing L, Huang H, Zhu L, et al.: Evaluation of Plasma Endothelial Microparticles in Pre-Eclampsia. J. Int. Med. Res. 2014; 42(1): 42â51. PubMed Abstract | Publisher Full Text\n\nMuralidharan-Chari V, Clancy JW, Sedgwick A, et al.: Microvesicles: Mediators of Extracellular Communication during Cancer Progression. J. Cell Sci. 2010; 123(10): 1603â1611. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBattistelli M, Falcieri E: Apoptotic Bodies: Particular Extracellular Vesicles Involved in Intercellular Communication. Biology (Basel). 2020; 9(1) PubMed Abstract | Publisher Full Text | Free Full Text\n\nKwok ZH, Wang C, Jin Y: Extracellular Vesicle Transportation and Uptake by Recipient Cells: A Process to Regulate Human Diseases. Processes (Basel). 2021; 9(2): 1â16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTkach M, Kowal J, Zucchetti AE, et al.: Qualitative Differences in T-Cell Activation by Dendritic Cell-Derived Extracellular Vesicle Subtypes. EMBO J. 2017; 36(20): 3012â3028. 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PubMed Abstract | Publisher Full Text\n\nVerweij FJ, Bebelman MP, Jimenez CR, et al.: Correction: Quantifying Exosome Secretion from Single Cells Reveals a Modulatory Role for GPCR Signaling. J. Cell Biol. 2018; 217(3): 1157â1157. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEscrevente C, Keller S, Altevogt P, et al.: Interaction and Uptake of Exosomes by Ovarian Cancer Cells. BMC Cancer. 2011; 11(1): 1â10. Publisher Full Text\n\nEguchi S, Takefuji M, Sakaguchi T, et al.: Cardiomyocytes Capture Stem Cell-Derived, Anti-Apoptotic MicroRNA-214 via Clathrin-Mediated Endocytosis in Acute Myocardial Infarction. J. Biol. Chem. 2019; 294(31): 11665â11674. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBenmerah A, Bayrou M, Cerf-Bensussan N, et al.: Inhibition of Clathrin-Coated Pit Assembly by an Eps15 Mutant. J. Cell Sci. 1999; 112(9): 1303â1311. Publisher Full Text\n\nFeng D, Zhao WL, Ye YY, et al.: Cellular Internalization of Exosomes Occurs through Phagocytosis. Traffic. 2010; 11(5): 675â687. PubMed Abstract | Publisher Full Text\n\nBarrÚs C, Blanc L, Bette-Bobillo P, et al.: Galectin-5 Is Bound onto the Surface of Rat Reticulocyte Exosomes and Modulates Vesicle Uptake by Macrophages. Blood. 2010; 115(3): 696â705. PubMed Abstract | Publisher Full Text\n\nGonda A, Moyron R, Kabagwira J, et al.: Cellular-Defined Microenvironmental Internalization of Exosomes. Extracellular Vesicles and Their Importance in Human Health. 2019. Publisher Full Text\n\nParton RG, McMahon KA, Wu Y: Caveolae: Formation, Dynamics, and Function. Curr. Opin. Cell Biol. 2020; 65: 8â16. Publisher Full Text\n\nCosta Verdera H, Gitz-Francois JJ, Schiffelers RM, et al.: Cellular Uptake of Extracellular Vesicles Is Mediated by Clathrin-Independent Endocytosis and Macropinocytosis. J. Control. Release. 2017; 266: 100â108. PubMed Abstract | Publisher Full Text\n\nKerr MC, Teasdale RD: Defining Macropinocytosis. Traffic. 2009; 10(4): 364â371. 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PubMed Abstract | Publisher Full Text\n\nXue J, Liu Y, Luo F, et al.: Circ100284, via MiR-217 Regulation of EZH2, Is Involved in the Arsenite-Accelerated Cell Cycle of Human Keratinocytes in Carcinogenesis. Biochim. Biophys. Acta (BBA) - Mol. Basis Dis. 2017; 1863(3): 753â763. PubMed Abstract | Publisher Full Text\n\nTian L, Cao J, Jiao H, et al.: CircRASSF2 Promotes Laryngeal Squamous Cell Carcinoma Progression by Regulating the MiR-302b-3p/IGF-1R Axis. Clin. Sci. 2019; 133(9): 1053â1066. PubMed Abstract | Publisher Full Text\n\nPavlyukov MS, Yu H, Bastola S, et al.: Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma via Intercellular Transfer of Splicing Factors. Cancer Cell. 2018; 34(1): 119â135.e10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAilawadi S, Wang X, Gu H, et al.: Pathologic Function and Therapeutic Potential of Exosomes in Cardiovascular Disease. Biochim. Biophys. Acta. 2015; 1852(1): 1â11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBang C, Batkai S, Dangwal S, et al.: Cardiac Fibroblast-Derived MicroRNA Passenger Strand-Enriched Exosomes Mediate Cardiomyocyte Hypertrophy. J. Clin. Invest. 2014; 124(5): 2136â2146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRibeiro MF, Zhu H, Millard RW, et al.: Exosomes Function in Pro- and Anti-Angiogenesis. Curr Angiogenes. 2013; 2(1): 54â59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang Y, Hu YW, Zheng L, et al.: Characteristics and Roles of Exosomes in Cardiovascular Disease. DNA Cell Biol. 2017; 36(3): 202â211. Publisher Full Text\n\nHowitt J, Hill AF: Exosomes in the Pathology of Neurodegenerative Diseases. J. Biol. Chem. 2016; 291(52): 26589â26597. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSamanta S, Rajasingh S, Drosos N, et al.: Exosomes: New Molecular Targets of Diseases. Acta Pharmacol. Sin. 2018; 39(4): 501â513. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu H, Chen L, Peng Y, et al.: Dendritic Cells Loaded with Tumor Derived Exosomes for Cancer Immunotherapy. Oncotarget. 2018; 9(2): 2887â2894. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGilligan KE, Dwyer RM: Engineering Exosomes for Cancer Therapy. Int. J. Mol. Sci. 2017; 18(6) PubMed Abstract | Publisher Full Text | Free Full Text\n\nGomari H, Moghadam MF, Soleimani M: Targeted Cancer Therapy Using Engineered Exosome as a Natural Drug Delivery Vehicle. Onco. Targets. Ther. 2018; 11: 5753â5762. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen DS, Mellman I: Oncology Meets Immunology: The Cancer-Immunity Cycle. Immunity. 2013; 39(1): 1â10. PubMed Abstract | Publisher Full Text\n\nPitt JM, André F, Amigorena S, et al.: Dendritic Cell-Derived Exosomes for Cancer Therapy. J. Clin. Invest. 2016; 126(4): 1224â1232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZitvogel L, Regnault A, Lozier A, et al.: Eradication of Established Murine Tumors Using a Novel Cell-Free Vaccine: Dendritic Cell Derived Exosomes. Nat. Med. 1998; 4(5): 594â600. PubMed Abstract | Publisher Full Text\n\nMunich S, Sobo-Vujanovic A, Buchser WJ, et al.: Dendritic Cell Exosomes Directly Kill Tumor Cells and Activate Natural Killer Cells via TNF Superfamily Ligands. Oncoimmunology. 2012; 1(7): 1074â1083. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBobrie A, Krumeich S, Reyal F, et al.: Rab27a Supports Exosome-Dependent and -Independent Mechanisms That Modify the Tumor Microenvironment and Can Promote Tumor Progression. Cancer Res. 2012; 72(19): 4920â4930. Publisher Full Text\n\nMarleau AM, Chen CS, Joyce JA, et al.: Exosome Removal as a Therapeutic Adjuvant in Cancer. J. Transl. Med. 2012; 10(1): 1â12. Publisher Full Text\n\nvon Schulze A , Deng F: A Review on Exosome-Based Cancer Therapy. J. Cancer Metastasis Treat. 2020; 2020. Publisher Full Text\n\nDaÃler-Plenker J, KÃŒttner V, Egeblad M: Communication in Tiny Packages: Exosomes as Means of Tumor-Stroma Communication. Biochim. Biophys. Acta Rev. Cancer. 2020; 1873(2): 188340. PubMed Abstract | Publisher Full Text\n\nHannafon BN, Ding WQ: Intercellular Communication by Exosome-Derived MicroRNAs in Cancer. Int. J. Mol. Sci. 2013; 14(7): 14240â14269. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeng F, Miller J: A Review on Protein Markers of Exosome from Different Bio-Resources and the Antibodies Used for Characterization. J. Histotechnol. 2019; 42(4): 226â239. PubMed Abstract | Publisher Full Text\n\nDeng F, Magee N, Zhang Y: Decoding the Role of Extracellular Vesicles in Liver Diseases. Liver Res. 2017; 1(3): 147â155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBruschi M, Ravera S, Santucci L, et al.: The Human Urinary Exosome as a Potential Metabolic Effector Cargo. Expert Rev. Proteomics. 2015; 12(4): 425â432. Publisher Full Text\n\nKatakowski M, Buller B, Zheng X, et al.: Exosomes from Marrow Stromal Cells Expressing MiR-146b Inhibit Glioma Growth. Cancer Lett. 2013; 335(1): 201â204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCho JA, Yeo DJ, Son HY, et al.: Exosomes: A New Delivery System for Tumor Antigens in Cancer Immunotherapy. Int. J. Cancer. 2005; 114(4): 613â622. Publisher Full Text\n\nHall J, Prabhakar S, Balaj L, et al.: Delivery of Therapeutic Proteins via Extracellular Vesicles: Review and Potential Treatments for Parkinsonâs Disease, Glioma, and Schwannoma. Cell. Mol. Neurobiol. 2016; 36(3): 417â427. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMalhotra H, Sheokand N, Kumar S, et al.: Exosomes: Tunable Nano Vehicles for Macromolecular Delivery of Transferrin and Lactoferrin to Specific Intracellular Compartment. J. Biomed. Nanotechnol. 2016; 12(5): 1101â1114. Publisher Full Text\n\nLai RC, Tan SS, Teh BJ, et al.: Proteolytic Potential of the MSC Exosome Proteome: Implications for an Exosome-Mediated Delivery of Therapeutic Proteasome. Int J Proteomics. 2012; 2012: 1â14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHermans IF, Ritchie DS, Yang J, et al.: CD8+ T Cell-Dependent Elimination of Dendritic Cells in vivo Limits the Induction of Antitumor Immunity. J. Immunol. 2000; 164(6): 3095â3101. Publisher Full Text\n\nLuketic L, Delanghe J, Sobol PT, et al.: Antigen Presentation by Exosomes Released from Peptide-Pulsed Dendritic Cells Is Not Suppressed by the Presence of Active CTL. J. Immunol. 2007; 179(8): 5024â5032. Publisher Full Text\n\nAspe JR, Wall NR: Survivin-T34A: Molecular Mechanism and Therapeutic Potential. Onco. Targets. Ther. 2010; 3: 247â254. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaari H, Lázaro-Ibáñez E, Viitala T, et al.: Microvesicle- and Exosome-Mediated Drug Delivery Enhances the Cytotoxicity of Paclitaxel in Autologous Prostate Cancer Cells. J. Control. Release. 2015; 220(Pt B): 727â737. PubMed Abstract | Publisher Full Text\n\nSpector PE, Fox S, Penney LM, et al.: The Dimensionality of Counterproductivity: Are All Counterproductive Behaviors Created Equal?. J. Vocat. Behav. 2006; 68(3): 446â460. Publisher Full Text\n\nUpadhya R, Shetty AK: Extracellular Vesicles for the Diagnosis and Treatment of Parkinsonâs Disease. Aging Dis. 2021; 12(6): 1438â1450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu H, Yang X, Xiao X, et al.: Human Adipose Mesenchymal Stem Cell-Derived Exosomes Protect Mice from DSS-Induced Inflammatory Bowel Disease by Promoting Intestinal-Stem-Cell and Epithelial Regeneration. Aging Dis. 2021; 12(6): 1423â1437. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeng W, He C, Hao Y, et al.: Prospects and Challenges of Extracellular Vesicle-Based Drug Delivery System: Considering Cell Source. Drug Deliv. 2020; 27(1): 585â598. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang X, Liu D, Gao Y, et al.: The Biology and Function of Extracellular Vesicles in Cancer Development. Front. Cell Dev. Biol. 2021; 9: 3192. Publisher Full Text\n\nHong EJ, Choi DG, Shim MS: Targeted and Effective Photodynamic Therapy for Cancer Using Functionalized Nanomaterials. Acta Pharm. Sin. B. 2016; 6(4): 297â307. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKawikova I, Askenase PW: Diagnostic and Therapeutic Potentials of Exosomes in CNS Diseases. Brain Res. 2015; 1617: 63â71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi X, Tsibouklis J, Weng T, et al.: Nano Carriers for Drug Transport across the Blood-Brain Barrier. J. Drug Target. 2017; 25(1): 17â28. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "204660",
"date": "15 Sep 2023",
"name": "Raghavendra Babu Y. P.",
"expertise": [
"Reviewer Expertise i Have been involved in Forensic Medicine teaching since last 18 years"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have delved on reviewing the literature with regards to EVs as a novel way of delivering drugs in cancer and other diseases.\nThe manuscript is lucidly written with wide range of issues related to EVs and their physiology, utility and probable ways to utilise them as drug delivery systems. The review gives factual information about EVs and delves throughly into the current topic of EVs.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
},
{
"id": "204580",
"date": "20 Oct 2023",
"name": "Charan Kishor Shetty",
"expertise": [
"Reviewer Expertise Forensic Pathology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBakkannavar and co-authors investigated the topic of Extracellular vesicles (EVs) as a novel drug delivery system to target cancer and other diseases. They specifically focused on the hypothesis that EVs are regarded as potent carriers of therapeutics that can be used as a therapeutic agent for diseases like cancer.\nThe authors have a very well-organized Introduction by elaborating biological functions of Extracellular vesicles, pathogenesis, and their role in cancer treatment. Moreover, they have described how EVs are derived from immune cells to suppress the growth of cancer cells, inhibition of cancer-derived EVs release, EVs as carriers of genes, and finally EVs as carriers of chemotherapeutic drugs and their use in treatment of other diseases. The article also gives us an insight into the challenges of EVs in drug delivery.\nThe manuscript is certainly well-written and attractive. Lastly, I think that this article conveys the message that EVs offer novel and important tools in cancer treatment.\nIt would be great if the authors could add any ongoing EV carrier drugs in use or under production.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-329
|
https://f1000research.com/articles/12-327/v1
|
23 Mar 23
|
{
"type": "Software Tool Article",
"title": "AmpSeqR: an R package for amplicon deep sequencing data analysis",
"authors": [
"Jiru Han",
"Jacob E. Munro",
"Melanie Bahlo",
"Jiru Han",
"Jacob E. Munro"
],
"abstract": "Amplicon sequencing (AmpSeq) is a methodology that targets specific genomic regions of interest for polymerase chain reaction (PCR) amplification so that they can be sequenced to a high depth of coverage. Amplicons are typically chosen to be highly polymorphic, usually with several highly informative, high frequency single nucleotide polymorphisms (SNPs) segregating in an amplicon of 100â200 base pair (bp). This allows high sensitivity detection and quantification of the frequency of each sequence within each sample making it suitable for applications such as low frequency somatic mosaicism detection or minor clone detection in mixed samples. AmpSeq is being increasingly applied to both biological and medical studies, in applications such as cancer, infectious diseases and brain mosaicism studies. Current bioinformatics pipelines for AmpSeq data processing lack downstream analysis, have difficulty distinguishing between true sequences and PCR sequencing errors and artifacts, and often require bioinformatic expertise. We present a new R package: AmpSeqR, designed for the processing of deep short-read amplicon sequencing data, with a focus on infectious diseases. The pipeline integrates several existing R packages combining them with newly developed functions to perform optimal filtering of reads to remove noise and improve the accuracy of the detected sequences data, permitting detection of very low frequency clones in mixed samples. The package provides useful functions including data pre-processing, amplicon sequence variants (ASVs) estimation, data post-processing, data visualization, and automatically generates a comprehensive Rmarkdown report that contains all essential results facilitating easy inclusion into reports and publications. AmpSeqR is publicly available at https://github.com/bahlolab/AmpSeqR.",
"keywords": [
"amplicon sequencing",
"data visualization",
"summary report",
"R package"
],
"content": "Introduction\n\nAmplicon sequencing (AmpSeq) is increasingly used in biological and medical studies for its high depth of coverage, allowing identification of rare variants, and also the ability to multiplex hundreds of samples in a single run, thereby reducing the sequencing costs. AmpSeq is highly scalable and cheap, permitting low-cost data generation for large sample sizes. It can also be multiplexed to further increase resolution. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), continues to pose a serious threat to the global population health. AmpSeq has been widely used to target the SARS-CoV-2 viral genome regions to monitor the presence of SARS-CoV-2 and detect the emerging variants.1,2 AmpSeq methods are also widely used in infectious disease characterization and surveillance studies (e.g., malaria infection) to monitor the emergence and spread of drug resistance,3â7 to examine population structure,8,9 relapse,10 to estimate clearance rates11 and to track within-host dynamics in longitudinal studies.12\n\nThere are several bioinformatics pipelines for processing AmpSeq data, such as DADA2,13 SeekDeep,14 and HaplotypR.15 SeekDeep and HaplotypR were specifically developed and tailored to study Plasmodium spp. However, these pipelines have some limitations, such as a lack of downstream analysis, difficulty in distinguishing between true sequence and PCR sequencing errors and artifacts, and often requiring extensive bioinformatics expertise. We introduce AmpSeqR, a freely available R package (https://github.com/bahlolab/AmpSeqR) that performs AmpSeq data analysis. The pipeline integrates several R packages as well as newly developed functions to filter out sequencing noise and improve the accuracy of the detected sequence data. The pipeline offers various analysis steps including data pre-processing, amplicon sequence variants (ASVs) estimation, data post-processing, data visualization, and automatically generates a comprehensive report in Rmarkdown that contains all essential results. This pipeline is designed to simplify bioinformatics processing leading to a comprehensive pipeline that starts from raw FASTQ files and finishes by generating a final reproducible report, from a reproducible pipeline. We apply AmpSeqR to various AmpSeq datasets, including the SARS-CoV-2 dataset and malaria parasite Plasmodium falciparum datasets.\n\n\nMethods\n\nThe AmpSeqR package was built in R and is hosted on GitHub. It can be installed from https://github.com/bahlolab/AmpSeqR. The input files for AmpSeqR are the standard paired-end FASTQ format provided by the common Illumina sequencing platforms (e.g., MiSeq), as well as sample barcodes and target amplicon details.\n\nAmpSeqR was developed and tested on R (version 4.1.3) with several other dependencies and the base functions in R and is compatible with Mac OS X, Windows, and major Linux operating systems. AmpSeqR is maintained at GitHub (https://github.com/bahlolab/AmpSeqR). The archived source code can be found in https://doi.org/10.5281/zenodo.7580184.16 AmpSeqR uses Biostrings17 (v2.62.0, RRID:SCR_016949), ShortRead18 (v1.52.0, RRID:SCR_006813), DADA213 (v1.20.0), DECIPHER19 (v2.20.0, RRID:SCR_006552), rlang (v1.0.2), Rmarkdown (v2.13), gtools (v3.9.2), withr (v2.5.0), utils (v4.1.1), cowplot (v1.1.1), furrr (v0.2.3), future (v1.24.0), GenomicRanges20 (v1.46.1), VariantAnnotation21 (v1.38.0), S4Vectors (v0.32.3), IRanges20 (v2.28.0), plyr (v1.8.7), DT (v0.22), plotly (v4.10.0, RRID:SCR_013991), viridisLite (v0.4.0), flextable (v0.7.0), scales (v1.2.0, RRID:SCR_019295), magrittr (v2.0.3), heatmaply (v1.3.0), digest (v0.6.29), tidyselect (v1.1.2), data.table (v1.14.2), ComplexHeatmap22 (v2.11.1, RRID:SCR_017270), htmltools (v0.5.2), ape (v5.6-2, RRID:SCR_017343), randomcoloR (v1.1.0.1), ggtree23 (v3.0.4, RRID:SCR_018560), ggstance (v0.3.5), tibble (v3.1.8), stats (v4.1.3) and several tidyverse packages (v1.3.1, RRID:SCR_019186). The workflow is illustrated in Figure 1.\n\nData pre-processing\n\nThe data pre-processing step demultiplexes the raw paired-end FASTQ reads and trims the sample barcodes and target amplicon primer sequences as well as assigning the sequence to each sample and marker (amplicon target) combination. As each individual has a unique oligonucleotide barcode and each marker has a unique primer sequence distinguishing it from other markers, sequence reads can be de-multiplexed to separate individual samples and different markers, followed by trimming the primer sequence. The data pre-processing is handled using the Biostrings, ShortRead, rlang, magrittr, future, and tidyverse R packages.\n\nAmplicon sequence variants (ASVs) estimation\n\nThe amplicon sequence variants (ASVs) estimation process identifies amplicon haplotype sequences. We leveraged several functions in the DADA2 R package, such as filterAndTrim, derepFastq, learnErrors, dada, and mergePairs to obtain the haplotype sequence. DADA2 can handle sequences with indels and accurately determines sequence variants leading us to choose DADA2 as the preprocessing tool to call ASVs in our AmpSeqR pipeline. For merging of paired-end reads we provided an option that can process the paired-end reads without any overlap. We also added some functions based on ShortRead, future, and tidyverse R packages for automatic downsampling of each sample/amplicon combination to a certain number of reads (e.g., 10,000) to reduce the computation time required. This step reduces excessive coverage and computational cost. The GATK (RRID:SCR_001876) documentation states that having additional data is not informative when read coverage exceeds a certain depth. A coverage of 10,000 reads was found to be sufficient to detect minor clones with a frequency as low as 0.1%.15\n\nData post-processing\n\nThis step primarily optimises amplicon haplotype calling through chimeric read detection and removal and variant filtration. The removal of sequencing background noise such as ultra-rare variants and chimeric reads generated during PCR amplification are important steps for the generation of high fidelity amplicon sequencing data. Chimeric reads are sequences formed from two or more biologically distinct sequences joined together during PCR amplification. Chimeric reads are much more frequent in amplicon sequencing applications in comparison to whole-exome sequencing (WES) or whole genome sequencing (WGS) because the same as well as very similar sequences appear in increased abundance.24 In addition, there are several types of indel errors in short-read Illumina sequencing data, such as homopolymers and terminal indels, which are the main source of low-quality indel calls.25 Several parameters are used to filter haplotypes, such as: (i) haplotype frequency, (ii) sequence similarity (haplotype sequences were mapped against reference sequence for each amplicon marker to calculate the sequence similarity), as well as (iii) variant heterozygosity, and (iv) minor allele frequency (MAF) in a given dataset. The detailed parameter options and their default values are listed in Table 1. After removing the background noise, the final haplotypes for each sample and marker combination are identified. This step is handled using the Biostrings, DECIPHER, rlang, withr, GenomicRanges20 (RRID:SCR_000025), VariantAnnotation21 (RRID:SCR_000074), S4Vectors, IRanges20 (RRID:SCR_006420), Parallel, gtools, utils, magrittr, tibble, and tidyverse R packages.\n\nData visualization\n\nThe data visualization step generates a comprehensive Rmarkdown report that contains various summary data visualizations and data summaries, such as quality checks, performed at both the individual/sample and amplicon level, amplicon haplotype sequence counts, haplotype diversity metrics of amplicon markers in the dataset, haplotype sequence visualization, haplotype tree generation, and principal component analysis (PCA). Quality checks include checking sample and amplicon marker information, an overview of read counts to track reads at various points in the pipeline, missing values in the dataset, and passed samples and markers. Haplotype diversity metrics such as expected heterozygosity (He), number of single nucleotide polymorphisms (SNPs), and number of unique haplotypes detected per sample per marker, are generated to help explore the amplicon marker diversity in the dataset. Detected haplotype sequences can be aligned to the reference genome and visualized to explore SNP positions and nucleotide changes. The haplotype tree is created by integrating all major haplotype sequences (>50% relative abundance) in each sample and performing multiple sequence alignment to identify the similarity between samples. PCA is also performed to visualize the sample structure. The process is handled using the Biostrings, Rmarkdown, plyr, DT, plotly, viridisLite, stats, flextable, ComplexHeatmap,22 scales, heatmaply, digest, tidyselect, data.table, DECIPHER, htmltools, pheatmap (RRID:SCR_016418), ape, randomcoloR, ggtree,23 ggstance, and several tidyverse R packages.\n\nThe major functions of the AmpSeqR package are summarised in Table 2.\n\n\nUse cases\n\nIn this section, we demonstrate the application of AmpSeqR for the data referenced in Datasets.\n\nThree datasets are used for the Use cases section. Dataset 1 is provided with the package and datasets 2 and 3 need to be downloaded via the published papers in which they appeared.\n\nDataset 1: Synthetic Plasmodium falciparum AmpSeq data\n\nThe synthetic P. falciparum paired-end amplicon sequencing data (FASTQ) was generated using the ART26 and ShortRead18 packages. The dataset consists of five P. falciparum samples with defined mixtures (1:1, 1:10, 1:100, 1:500, 1:1000) of two P. falciparum reference genomes Pf3D7 and PfDd2 (downloaded from PlasmoDB). We selected two amplicon markers, the P. falciparum surface marker genes circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). The FASTQ sequence data, as well as additional amplicon primer and sample barcode details are included in the AmpSeqR package.\n\nDataset 2: SARS-CoV-2 AmpSeq data\n\nThe dataset for multiplexed SARS-CoV-2 was obtained through the Aynaud et al.1 study. We downloaded the PoC cohort, which includes 19 nasopharyngeal swab samples, of which 17 were positive for SARS-CoV-2. This study targeted six amplicon markers: one human gene Peptidylprolyl Isomerase B (PPIB) as a quality control, and five SARS-CoV-2 regions targeting the Nucleocapsid (N), Envelope (E), RNA-dependent RNA polymerase (RdRP), and two regions of the Spike (S) gene that correspond to the receptor-binding domain (RBD) and the polybasic cleavage site (PBS). Amplicon primer details are given in Aynaud et al.1 Sequencing was performed using the Illumina MiSeq sequencing platform. This dataset and metadata are available in Gene Expression Omnibus (GEO) at NCBI (GEO accession number GSE160031). The complete genome SARS-CoV-2 isolate Wuhan-Hu-1 (NC_045512) was used as the reference genome for sequence analysis and was downloaded from NCBI (NC_045512.2).\n\nDataset 3: Plasmodium falciparum AmpSeq data\n\nThe P. falciparum dataset can be downloaded from the Lerch et al.15 study. The dataset consists of 16 defined mixtures of P. falciparum genomic DNA samples, where the mixtures of two P. falciparum lab strains HB3 and 3D7 were combined in various proportions (1:1, 1:10, 1:50, 1:100, 1:500, 1:1000, 1:1500, and 1:3000). Lerch et al. targeted two P. falciparum surface marker genes: (i) the conserved Plasmodium membrane protein (CPMP) and, (ii) the circumsporozite protein (CSP). This dataset is available on the NCBI-SRA website: BioProject accession PRJNA381546. Amplicon primer and sample barcodes details are given in the Lerch et al. study and can be downloaded from https://github.com/lerch-a/HaplotypR.git. This provides a test dataset to evaluate the detectability of minority clones of AmpseqR.\n\nThe AmpSeqR package is hosted in GitHub and can be installed with the following command line.\n\nLoad the AmpSeqR package into the workspace as well as other packages required for the following analysis.\n\nHere we present the AmpSeqR workflow with a minimal example dataset (example_data). The input files are the standard paired-end FASTQ files provided by the Illumina sequencing platforms, as well as sample barcodes and target amplicon details. The sample barcodes file should include sample_id, barcode_fwd, barcode_rev, sample (sample name, can be the same as sample_id), info (e.g., sample type). The target amplicon detail file should include marker_id, primer_fwd, primer_rev, seq (reference sequence), chrom (chromosome), start (reference sequence start position), end (reference sequence end position). This file should be set up in a text editor or excel and saved as text CSV format (*.csv) file.\n\nThis step demultiplexes the raw paired-end FASTQ reads and trims the sample barcodes and target amplicon primer sequences and assigns reads to each sample and each amplicon marker combination.\n\nHere, we first use the synthetic Plasmodium falciparum AmpSeq data.\n\nThe analysis directory is then created.\n\nNext, we demultiplex the reads using the demultiplex_reads function as follows.\n\nThe main outputs from the demultiplex_reads function are:\n\n⢠demultiplex folder: demultiplexed paired-end FASTQ files.\n\n⢠demultiplex.rds: the demultiplexed table in RDS format which includes sample_id, marker_id, reads_1 (the forward read FASTQ file path), reads_2 (the reverse read FASTQ file path), n (number of demultiplexed reads), sample, info.\n\nBefore filtering and trimming the paired-end FASTQ files we can visualize the quality profiles of the forward and reverse reads of different amplicon markers by using the plot_quality function in AmpSeqR which integrates several functions from the ShortRead, DADA2, cowplot and tidyverse packages (Figure 2).\n\nThe red line shows the mean quality score of all samples in each cycle. Color represents the proportion of reads of each quality score in each cycle.\n\nAccording to the quality profiles of the forward and reverse reads, we can define the trim position for the forward and reverse reads to remove low-quality bases. The marker_trim should be a data frame with three columns marker_id (character), trim_fwd (integer), and trim_rev (integer), or can be left unset (NULL, run without trimming).\n\nThe next step filters the poor-quality reads and trim low-quality bases as follows. Filtering is implemented using the dada_filter function, which uses the filterAndTrim function from the DADA2 package.\n\nThe main outputs for the dada_filter function are:\n\n⢠a filter folder: filtered and trimmed paired-end FASTQ files.\n\n⢠a filtered_reads.rds table: the filtered table in RDS format which includes sample_id, marker_id, n (number of demultiplexed reads), sample, info, reads_1 (the filtered and trimmed forward FASTQ file path), reads_2 (the filtered and trimmed reverse FASTQ file path), n_in (number of reads before filtering and trimming), n_out (number of reads after filtering and trimming).\n\nThe downsample_reads function randomly downsamples each sample dataset to a pre-specified number of reads (default, 10,000 reads) to reduce the computation time.\n\nThe main outputs for the downsample_reads function are:\n\n⢠the downsample folder: downsampled paired-end FASTQ files with a read number greater than n_sample.\n\n⢠the subsampled_reads.rds table: the downsampled table in RDS format includes sample_id, marker_id, sample, info, reads_1 (the downsampled forward FASTQ file path), reads_2 (the downsampled reverse FASTQ file path), n_out (number of reads before downsampling), n (number of reads after downsampling).\n\nThe dada_seq_tbl function use several functions in DADA2, Biostrings, DECIPHER, and Parallel packages, such as derepFastq, learnErrors, dada, mergePairs, AlignSeqs, DistanceMatrix to filter noise reads and merge the paired-end reads and obtain the amplicon haplotype sequence. When paired-end reads do not overlap, the min_overlap parameter should be set to -1.\n\nThe main outputs for the dada_seq_tbl function are:\n\n⢠the seq_tbl.rds table: the amplicon haplotype sequence table in RDS format includes sample_id, marker_id, sequence (haplotype sequence), count (read counts), status (initial haplotype status), sample, info.\n\nThe annotate_seq_tbl function leverages various useful functions in Biostrings and DECIPHER to determine the amplicon haplotype status and to distinguish the true haplotype sequences from sequencing background noise and artifacts, including ultra-rare variants and chimeric reads generated during PCR amplification.\n\nThe main outputs for the annotate_seq_tbl function are:\n\n⢠the seq_ann_tbl.rds table: the annotated amplicon haplotype sequence table in RDS format includes sample_id, marker_id, sequence (haplotype sequence), count (read counts), status (inferred haplotype status), sample, info, ident (sequence similarity), ident_z (standardized sequence similarity).\n\nThe amplicon sequence haplotype status includes the following states:\n\n⢠pass: the amplicon sequences haplotype has passed all quality control checks,\n\n⢠low_sample_count, failed: the sample read count lower than the minimum number of read per sample per marker,\n\n⢠low_asv_count, failed: the amplicon sequence haplotype counts lower than the minimum number of read for each haplotype,\n\n⢠low_asv_freq, failed: the amplicon sequence haplotype frequency lower than the minimum haplotype frequency,\n\n⢠low_ident, failed: the amplicon sequence haplotype similarity (after being mapped to the reference) is lower than the minimum sequence similarity,\n\n⢠low_ident_z, failed: the standardized amplicon sequence haplotype similarity (after being mapped to the reference) lower than the minimum standardized sequence similarity,\n\n⢠chimera, failed: the sequence is a chimera of reads,\n\n⢠multiple, failed: multiple fail types.\n\nThe sequence_filter function is mainly optimizing amplicon haplotype calling and uses several parameters, such as sample missingness, amplicon marker missingness, the number of alleles at a locus, as well as variant heterozygosity and MAF in a given dataset. This function is implemented with Biostrings, magrittr, withr, GenomicRanges, VariantAnnotation, S4Vectors, IRanges, and tidyverse R packages.\n\nThe main outputs for the sequence_filter function are:\n\n⢠the seq_flt_tbl.rds table: the final amplicon haplotype sequence table in RDS format which includes sample_id, marker_id, sequence (haplotype sequence), count (read counts), masked (TRUE indicates the sequence contains variants that might be errors, and replaces the nucleotide with the reference genome nucleotide), status (haplotype status, all pass), ident (sequence similarity), haplotype (a named panel of haplotypes for each marker), frequency (within-sample haplotype frequency for each marker), sample, info.\n\nThe generate_report function generates a HTML report including various summary data visualizations. The function is based on Biostrings, Rmarkdown, plyr, DT, plotly, viridisLite, stats, flextable, ComplexHeatmap, scales, heatmaply, digest, tidyselect, data.table, DECIPHER, htmltools, pheatmap, ape, randomcoloR, ggtree, ggstance, and tidyverse R packages. The HTML summary report for the synthetic P. falciparum dataset is available from Figshare (https://doi.org/10.6084/m9.figshare.21739121).27\n\nWe also include the process_run function that calls all the functions, making it easy to get all results.\n\nFor the SARS-CoV-2 AmpSeq data, the paired-end FASTQ data is already demultiplexed by sample. We first demultiplexed by marker and then run AmpSeqR from the filter and trim step as follows. Some output from the HTML report for the SARS-CoV-2 dataset is shown in Figure 3.\n\nA. The proportion of sequences that are finally retained. B. Missing values in the dataset. C. Haplotype diversity metrics for amplicon markers in the dataset. D. Read counts for amplicon markers and all samples in the dataset. E. Number of unique haplotypes detected per amplicon per sample. F. Visualisation of haplotype and reference sequences.\n\nThe detectability of the minor clone of the CPMP and CSP amplicon markers under different mixture ratios of P. falciparum is shown in Figure 4. The correct minor haplotype was detected in mixture proportions from 1:1 to 1:3000 in most mixtures and detected haplotype frequencies similar to the defined mixture proportion. These results demonstrate that AmpSeqR can detect low-frequency haplotypes accurately.\n\nThe x-axis represents the haplotype frequency, and the y-axis represents the sample. Colored by haplotype and labelled by haplotype and haplotype frequency. The grey bar represents a reference for different defined mixture proportions.\n\n\nConclusions\n\nWe present the AmpSeqR R package for analysis of AmpSeq data which was primarily developed for the analysis of infectious diseases. The pipeline offers various useful steps including data pre-processing, amplicon sequence variants (ASVs) estimation, data post-processing, data visualization and includes several parameters to filter noise reads and improve the accuracy of the detected haplotype. AmpSeqR allows users to easily analyze the AmpSeq data and generate an HTML report including various summary data visualizations. Additionally, the one-line commands for processing the data do not require extensive R knowledge which provides a user-friendly experience for R users of all levels of experience.",
"appendix": "Data availability\n\nFigshare: Underlying data for âAmpSeqR: an R package for amplicon deep sequencing data analysisâ. https://doi.org/10.6084/m9.figshare.21739121.v2. 27\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nNCBI GEO: A multiplexed, next-generation sequencing platform for high-throughput detection of SARS-CoV-2. Accession number GSE160031; https://identifiers.org/geo:GSE160031\n\nNCBI Genome: SARS-CoV-2 isolate Wuhan-Hu-1, complete genome. Accession number NC_045512; https://www.ncbi.nlm.nih.gov/nuccore/NC_045512.2\n\nBioProject: Amplicon deep sequencing of multi-clonal Plasmodium falciparum infections. Accession number PRJNA381546. https://identifiers.org/bioproject:PRJNA381546\n\n\nAcknowledgements\n\nThe authors would like to thank Shazia Ruybal (The Walter and Eliza Hall Institute of Medical Research), Javier Rosado Sandoval (Pasteur Institute), and Caitlin Bourke (The Walter and Eliza Hall Institute of Medical Research) for testing and feedback on the AmpSeqR package.\n\n\nReferences\n\nAynaud M-M, Hernandez JJ, Barutcu S, et al.: A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2. Nat. Commun. 2021; 12(1): 1405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYelagandula R, Bykov A, Vogt A, et al.: Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq. Nat. Commun. 2021; 12(1): 3132. 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}
|
[
{
"id": "319368",
"date": "09 Nov 2024",
"name": "Eduardo Castro-Nallar",
"expertise": [
"Reviewer Expertise My area of research is microbial genomics. I work primarily with environmental microbial communities understanding their ecology and evolution through a genetics/genomic lens. I have occasionally worked on software development that focuses on microbial genomics. I have also benchmarked tools developed for metagenomic and amplicon sequencing analysis."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe rationale for developing the new software tool is partly explained, as the value of amplicon sequencing and its importance in diverse scientific disciplines is clear. However, the rationale for developing this tool in the light of many other published tools with similar functionality is not clear.\nRegarding the performance. The manuscript does not mention compute time per sample or dataset so that it's difficult to assess whether someone should select this tool over another. Likewise, there's no comparative benchmark study with other published tools.\nIn summary, this is a wrapper tool that at its core uses DADA2 to infer amplicon sequence variants and offers some handy functions to conduct steps before and after DADA2's inference.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "330664",
"date": "12 Nov 2024",
"name": "Cristian Koepfli",
"expertise": [
"Reviewer Expertise Malaria epidemiology",
"diagnosis",
"and genomics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nJiru Han and colleagues describe a new bioinformatic pipeline for the analysis of amplicon sequencing data. The pipeline incorporates several existing bioinformatic tools and adds new ones, including a comprehensive visual report. One of the aims is to have a pipeline that is easier to use for non-bioinformaticians compared to other pipelines.\n\nAs a disclaimer to my review, I was closely involved in the development of HaplotypeR, and the generation of dataset 3 that was used in the current manuscript to test AmpSeqR. The code was tested by Gustavo da Silva, a student under my supervision.\nMain Comments: We were not able to run the code given in the manuscript in the conditions below.\nThe regular run option in AmpSeqR works as expected with its example files. However, when we applied it to our own aplicon sequencing samples for Plasmodium falciparum and Plasmodium vivax, it encountered an error during the demultiplexing step, where the program sorts the data. We formatted the files to match the example format exactly, but they still failed. We then tried different configurations, such as playing with the data frames and tables, but none of these attempts resolved the issue. Without clear guidance on how to structure non-example data, AmpSeqR's utility for processing diverse datasets is significantly limited. Additionally, the fast run option did not produce any output files for us. It fails even with the example files, and it also doesnât work with any of our custom samples - expected after the problems observed above. It would be helpful if the fast run generated output files at each step, rather than only at the final report stage, as this would make it easier to identify where problems might be occurring. Such files would be helpful for the regular run as well. Finally, when generating reports, AmpSeqR encounters an error in the âtrain()â function due to an unused argument, which may be linked to its interactions with ggplot2 or plotly. This error prevents the report from being created, leaving only the âseq_flt_tblâ table in the environment. It happens both with the regular run and the fast run.\n\nThe paper states that existing methods have âdifficulty in distinguishing between true sequence and PCR sequencing errors and artifactsâ. First, I am not sure what is meant by artifacts. The main challenge for any analysis of such types of sequencing typically is the distinction between PCR/sequencing errors and true minority clones present at low frequency within in sample. This is an inherent challenge to any bioinformatic analysis, it is not clear how AmpSeqR overcomes this issue. More discussion on what AmpSeqR does differently compared to other pipelines, and how this is achieved, would be useful. Either the introduction could be expanded, or a proper discussion section could be added.\n\nMinor comments: The writing is sometimes a bit confusing and could be clarified. E.g.:\n\nPlease clarify the following sentences: âand also the ability to multiplex hundreds of samples in a single run, thereby reducing the sequencing costs. AmpSeq is highly scalable and cheap, permitting low-cost data generation for large sample sizes. It can also be multiplexed to further increase resolution.â\nI believe the first âmultiplexedâ refers to multiplexing of samples, while the second refers to multiplexing of markers.\nI am a bit surprised about SARS-CoV-2 being highlighted in the introduction of an amplicon sequencing paper. A lot (though not all) of sequencing studies sequenced full genomes, which is different from amplicons of a few hundred bp. Likely, AmpSeqR is also useful for viral WGS data, but it would be good to discuss this more specifically. Just following the section on SARS-CoV-2, a sentence in the introduction states that âAmpSeq methods are also widely used in infectious disease characterization and surveillance studiesâ. Given covid-19 is an infectious disease, this sentence is confusing. Maybe change to âAmpSeq methods are widely used in characterization and surveillance studies of many other infectious diseasesâ. The meaning of the term relapse as used in P. vivax studies might not be known to readers. The term is used for different processes in different diseases (compare e.g. to cancer relapse).\n\nTypo: âwell as assigning the sequence to eachâ > assigns\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "333941",
"date": "22 Nov 2024",
"name": "Patrick Murigu Kamau Njage",
"expertise": [
"Reviewer Expertise Infectius disease microbial genomics and data analysis."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThough this is an amazing package that may solve analysis and reporting bottlenecks in amplicon sequencing data analysis, the newly developed functions that are time intensive may need to be benchmarked against existing tools in terms of computation time if this is possible.\n\nR packages are listed without a clear in the section \"Operation\" and \"Data visualization\" without a clear link to their roles. An appendix that lists the role of each of the R packages rather than just listing them in the text would make the package more reproducible.\nThe two sentences \"PCA is also performed to visualize the sample structure. The process is handled using the Biostrings, Rmarkdown, plyr, DT, plotly, viridisLite, stats, flextable, ComplexHeatmap,22 scales, heatmaply, digest, tidyselect, data.table, DECIPHER, htmltools, pheatmap (RRID:SCR_016418), ape, randomcoloR, ggtree,23 ggstance, and several tidyverse R packages.\" may deceptively imply that all the packages listed in the second sentence are for handling PCAs.\n\nFigure 4 caption needs to include a minimum amount of information for the reader to understand it without a deep search from the text. What are the mixture ratios?\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-327
|
https://f1000research.com/articles/12-325/v1
|
23 Mar 23
|
{
"type": "Research Article",
"title": "A cross-sectional study assessing family satisfaction in the intensive care environment in Bahrain: opportunities for improvement",
"authors": [
"Khadija Adel Hamed",
"Fatema Buzaid",
"Mais AlHafi",
"Jalal A. Alkhan",
"Khalid A. Ghaffar",
"Fatema Buzaid",
"Mais AlHafi",
"Jalal A. Alkhan",
"Khalid A. Ghaffar"
],
"abstract": "Background: Assessment of patientsâ family experience and levels of satisfaction have become a measure of intensive care. The aim of this study was to assess the needs of patientâs family members in an Intensive Care Unit (ICU) and to identify areas for improvement in quality care. Methods: A prospective, cross-sectional, a questionnaire survey study was carried out with 100 randomly selected family members of 77 critically ill patients cared for in surgical and medical ICUs. The family satisfaction (FS-ICU-24) questionnaire was given to family members, and the responses were used to assess their experience of ICU care. Results: The responses of patientsâ relatives exposed an overall level of satisfaction of 5.04 ± 1.104 on a scale of 1-6. Satisfaction with care was scored higher than satisfaction with decision-making. Family members reported low satisfaction with communication with physicians, the ICU atmosphere, and the waiting room atmosphere. Factors such as the completeness of the information received by relatives (p=0.007) and the ease of obtaining information (p=0.007) demonstrated a significant association with family satisfaction. Suggestions received from families with the high frequency where they need lengthier visiting hours, regular updates on medical reports, better care, and communication with ICU staff. Conclusions: Although overall family satisfaction was high, some areas emerged for improvement. The family members want to increase the length of visiting hours, frequency of patient status updates, communication with physicians, involvement in decision-making, and presence during medical visits. Considering the opportunity to improve ICU care, we propose periodic assessments of family satisfaction with ICU experiences.",
"keywords": [
"Intensive care unit",
"family satisfaction",
"family satisfaction-intensive care unit",
"health providers"
],
"content": "Introduction\n\nIn recent years, quality of care has become an issue of the highest priority in health care services, including aspects relating to the quality of life,1,2 care processes,3 and resource use.4 The ICU is the part of the hospital where seriously ill patients are cared for by specially trained staff. Along with them, family members also contribute to caring by providing support and resources to critically ill patients. Quality of care in the ICU includes the satisfaction of family members, who often take on responsibility for medical decisions on behalf of their ill person.5 Indeed, the critical care environment may place a prolonged burden on patients6,7 as well as their family members.8 It has been reported that the levels of satisfaction are somewhat lower in various aspects, particularly communication, emotional support, and the hospital environment.9\n\nThe healthcare strategy for one country cannot automatically be adopted by other countries. Hence, nationwide interest has developed regarding research to assess the needs of ICU patients, aiming to improve the quality of care in the ICU. However, patients in the ICU are often unable to communicate with the healthcare team during decision-making processes because of their illness or the effects of treatments, such as sedation and/or mechanical ventilation.10,11 In this regard, family members are not only visitors in the ICU, but, along with patients, they are also confronted with the complex process of ICU care, including a technical environment and frequent changeovers in staff.12,13 Physicians, nurses, and other health care providers in the ICU may find themselves caring not only for the physical needs of patients but also the psychological needs of the families.14,15 For these reasons, the experience of family members attending the ICU has been considered a major aspect while developing various tools for the assessment of family needs and satisfaction with ICU care.16 However, family satisfaction with care is a complex issue, and no gold standard definition is currently available to assess this concept.\n\nAlthough the first report of familiesâ opinions was published in the 1970s,17 systematic assessment of family satisfaction with intensive care was only possible after 2001, when the first measurable tool was developed by Wasser et al.,18 Subsequently, other tools were also developed, and validated. These tools are mostly questionnaires, but those are advocated internationally as means to improve patient care.\n\nAssessing the satisfaction of family members with the care and support they receive helps to improve the quality of care provided to families of ICU patients.19 Furthermore, improved communication with families of ICU patients has been shown to aid decision-making, resulting in reduced ICU stays, withdrawal of life-sustaining interventions when found appropriate,20,21 as well as increased overall psychological well-being.22 The American Society for Critical Care Medicine guidelines recommended that training in âgood communication skills should become a standard component of medical education for all ICU caregiversâ and further suggested that empathic communication, skilful discussion of prognosis, and effective shared decision-making reflected the quality of care provided in the ICU.23\n\nAlthough there are reports showing that family members are generally highly satisfied,12,24 several areas have been identified as opportunities for improvement, such as communication with ICU care providers, participation in patient care and decision-making,25 the ICU environment,26 collaboration between nursing staff and providers,27 and medical counselling for the attending family.28 Families often do not receive information about the status of patientsâ health even 48 hours after admission to the ICU.29 Moreover, as physicians are busy, they miss opportunities to listen, acknowledge and address the emotions of families.30\n\nThe growing evidence of literature suggests that the family needs in ICU vary in different parts of the world as it is affected by parameters relating to both patient and family members, as well as to ICU staff and infrastructure. This paper wants to understand the family needs in our ICU setup at Bahrain Defence Force Military Hospital. In order to improve the quality of ICU care, we must first measure our performance. Keeping this in mind, our study aimed to assess the levels of satisfaction of family members of patients admitted to the ICU and to identify the areas which need further improvement to upgrade the quality of ICU healthcare.\n\n\nMethods\n\nThe study protocol was reviewed and approved by the research ethics committee of Bahrain Defence Force Military Hospital (Registration No. 2019-511). The FS-ICU-24 survey was carried out in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki). Written informed consent was obtained from all participants.\n\nThis was a prospective questionnaire survey study designed to assess the levels of satisfaction of patientsâ families with the intensive care unit (ICU) at Bahrain Defence Force Military Hospital. We approached the relatives of ICU patients who attend visiting hours and chose the most frequent visitor. We recruited a maximum number of two first-degree relatives for each patient). Recruited family members included both immediate relatives (parents, siblings, spouses, and children) and extended family members (cousins, uncles, and friends) and those who visited patients at least twice during their ICU stay. A hard copy of the questionnaire and a copy of the consent sheet were distributed by KH and explained to them the purpose of the survey and about their voluntary participation in the survey. A total of 100 participants agreed to participate in the study within the data collection period. These participants were identified as relatives of 77 medical and surgical ICU patients. Most of the respondents submitted their responses on the same day, while the rest of them submitted them to the secretary who was not part of this survey within a given period of three weeks.\n\nAdults of both sex over the age of 18 who were relatives of patients admitted to the ICU for 48 hours or more between 1st August 2019 and 30th March 2022, who read and write Arabic, and who were willing to participate in the study were included. While the relatives of patients who were below 18 years of age, unable to read and write the Arabic language, and those who had not visited the patient at least twice during their stay were excluded from the study.\n\nFamily satisfaction was measured using the web-based FS-ICU-24 questionnaire with a few modifications that were essential as per our clinical set-up. This questionnaire assesses satisfaction in two domainsâsatisfaction with care and satisfaction with decision-making. At the start of the questionnaire, each participant had to provide their name, age, sex, and relationship to the patient and state whether they shared the same accommodation; the corresponding patient data were retrieved from the ICU records and electronic medical records.\n\nTo assess satisfaction with care, relatives were asked about the courtesy, respect, and compassion of the ICU staff. In addition, the following parameters were examined: assessment of pain; breathlessness and agitation; emotional support; teamwork; care for family; nurse communication; physician competence; doctor communication; ICU atmosphere; waiting room atmosphere; and frequency of communication. Furthermore, the satisfaction of the relatives with the information provided was measured by questions about their understanding of the information as well as its honesty, completeness, consistency, and ease with which it was obtained. Each question had five options (excellent, very good, good, fair, poor), of which one was selected.\n\nFurther details about inclusion in decision-making, participation in morning visits, time taken to assess concerns, control over care, and support in decision-making were obtained to evaluate the second part of the FS-ICU-24 (satisfaction with decision-making).\n\nContinuous variables were presented as means with standard deviation, while categorical variables were presented as frequencies and percentages. Depending on the data requirements, Mann-Whitney,31 Kruskal-Wallis,32 or Pearson correlations33 were used to test the association between baseline demographics and overall levels of family satisfaction. Regression analysis was used to determine which factors influenced overall family satisfaction in the ICU. SPSS software was used to conduct all analyses (version 26.0).34 A p-value of less than 0.05 was considered statistically significant.\n\n\nResults\n\nA total of 100 relatives of 77 ICU patients responded to the survey. Patient ages ranged from 19 to 92 years (mean age was 63.05 ± 8.27). Responses are summarized in Tables 1 and 2 as N (%) for each factor. Table 3 shows the association of baseline demographics with levels of family satisfaction in the ICU; 93.9% of respondents were immediately related to the patient and the remainder had an extended relationship. The study showed an 18.7% mortality rate. Neither Acute Physiology and Chronic Health Evaluation (Apache) scores (varying from 0 to 71, with a mean of 27.76 ± 18.624) nor the length of patient stays in the ICU (average 10 days) showed significant correlations with the level of family satisfaction. The overall level of family satisfaction was 5.04 ± 1.104 on a scale of 1â6, as illustrated in Figure 1. Table 4 shows the factors that have a statistically significant relationship with satisfaction levels.\n\n* Significant p-value <0.05, P-value was calculated by Regression analysis.\n\nAmong all the factors examined as shown in Table 4, two factors, namely receiving complete information from the patientâs relatives and the ease of getting information, demonstrated statistically significant relationships with the dependent variable. Since the coefficients of the significant factors are positive, we deduce that as the completeness of information about the patient and the ease with which it is obtained increases, so does the level of family satisfaction. With a coefficient of determination of 0.980, the model explained 98% of the variability of the independent variable (level of family satisfaction).\n\nThe most common comments and suggestions from patientsâ relatives were focused on the wish for lengthier visiting hours, for more visitors to be allowed to enter the ICU, and for a daily medical report about the patients, as shown in Figure 2. In addition, relatives wanted to be involved in making decisions and to be able to consult with doctors relatively frequently. Furthermore, 99% of respondents indicated a preference for being present during the morning medical visit.\n\n\nDiscussion\n\nFamily satisfaction is a key performance measure for assessing the quality of healthcare delivery25 and implementing quality developments.35 Family satisfaction is mainly based on the need for care, support, comfort assurance, courtesy, respect, and compassion, all of which are included in the FS-ICU 24 questionnaire. Moreover, FS-ICU 24 has been used in several previous studies and proved to be a reliable and validated tool for the assessment of family experience in the ICU.36,37\n\nIn this study, the questionnaire was given to 100 family members, all of whom submitted their written replies; this demonstrates that the need for assurance was met. Family members need to be able to trust caregivers and believe that their loved ones are receiving a good level of care, even if they are not present.38 In the context of our study, this means that the families of the patients should have been able to feel confident that giving adverse replies to the questionnaire would not influence the treatment and care given to their respective patients.\n\nIn this study, even though overall levels of satisfaction appeared with high percentages, relevant shortcomings still existed, leaving areas for improvement. We attempted to identify such areas of interest and opportunities for improvement. Satisfaction is a complex emotion, influenced by the gap between expectation and perception.25 Satisfaction with the ICU is composed of different parameters; however, two of these are commonly discussed, namely care and decision-making.5 In this study, families reported overall satisfaction with parameters relating to the ICU itself and to the patientâs care. The overall FS-ICU scores were relatively high for parameters such as compassion and respect are given to the patient, pain assessment, breathlessness assessment, attention to needs, emotional support, teamwork, care for family, nurse communication, and physician competence which go along with previous reports.12 However, the scores were lower than those obtained in studies conducted in different parts of the world such as the Canada,39 United Kingdom,40 and Germany.41 Moreover, in this study, the families were least satisfied with the waiting room atmosphere, physiciansâ communication, and the frequency of communication. This demonstrates that still there are opportunities to further increase family satisfaction by improving the quality of these parameters.\n\nIt is clear from the results that family members wanted physicians to be available for regular discussions in order to obtain information about their relativeâs medical condition and prognosis. However, this could be difficult for physicians because they are occupied with taking care of critically ill patients. Generally, nurses play a significant role in coordinating the flow of information between physicians and family members. It has been proposed that effective communication by physicians, skilful discussion of prognosis, and effective shared decision-making are the key elements for quality of care in the ICU.42 Importantly, effective communication by ICU staff can improve familiesâ understanding of the situation and reduce their psychological burden.43\n\nBecause of the nature of this study, there were time lags between families visiting the ICU and responding to the questionnaires rating their satisfaction. This raises the possibility that their replies regarding their experience in the ICU may have changed in the intervening period. Furthermore, many study limitations are restricted to drawing definite conclusions about the findings. For example, this study was conducted using a relatively small sample size and a homogenous population. As a result, study findings may not be generalized to other populations. In addition, the design of the study limits its potential use as a source of comparative information as it is a single centre, limited to ICUs and the processes of caregiving are likely to be the same. Furthermore, cultural, and economic factors were not considered in the questionnaire, although the evidence suggests that these factors can influence familiesâ responses.44 To overcome the above limitations, we propose further research with a larger sample size and a multi-centre and multi-ICU design in order to improve family satisfaction with ICU care, infrastructure, and the skills and competence of ICU employees.\n\n\nConclusions\n\nAlthough overall family satisfaction was high, some areas emerged for improvements, such as the length of visiting hours, frequency of patient status updates, communication with physicians, involvement in decision-making, and presence during medical visits. With this in mind, we propose that periodic assessment of family satisfaction with ICU experience should be carried out as it offers a valuable opportunity to improve the quality of care provided in the ICU.",
"appendix": "Data availability\n\nFigshare: Assessment of family satisfaction in the intensive care environment: opportunities for improvement, https://doi.org/10.6084/m9.figshare.21508710.v2. 45\n\nThis project contains the following underlying data:\n\n⢠Study data.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nThe authors wish to acknowledge Sharada Sawant for her contribution to data interpretation, manuscript writing, and reviewing and Shayma Alaamer for her contribution to statistical data analysis, Crown Prince Centre for Training and Medical Research, Bahrain Defence Force Royal Medical Services. The authors also thank all the participants.\n\n\nReferences\n\nHerridge MS, Cheung AM, Tansey CM, et al.: One-year outcomes in survivors of the acute respiratory distress syndrome. N. Engl. J. Med. 2003; 348: 683â693. Publisher Full Text\n\nGranja C, Lopes A, Moreira S, et al.: Patients' recollections of experiences in the intensive care unit may affect their quality of life. Crit. Care. 2005 Apr; 9(2): R96âR109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTerblanche M, Adhikari NK: The evolution of intensive care unit performance assessment. J. Crit. Care. 2006 Mar 1; 21(1): 19â22. Publisher Full Text\n\nRothen HU, Stricker K, Einfalt J, et al.: Variability in outcome and resource use in intensive care units. Intensive Care Med. 2007 Aug; 33(8): 1329â1336. Publisher Full Text\n\nDavidson JE, Powers K, Hedayat KM, et al.: Clinical practice guidelines for support of the family in the patient-centered intensive care unit: American College of Critical Care Medicine Task Force 2004â2005. Crit. Care Med. 2007 Feb 1; 35(2): 605â622. PubMed Abstract | Publisher Full Text\n\nJones C, BÀckman C, Capuzzo M, et al.: Precipitants of post-traumatic stress disorder following intensive care: a hypothesis generating study of diversity in care. Intensive Care Med. 2007 Jun; 33(6): 978â985. PubMed Abstract | Publisher Full Text\n\nBoer KR, van Ruler O , van Emmerik AA , et al.: Factors associated with posttraumatic stress symptoms in a prospective cohort of patients after abdominal sepsis: a nomogram. Intensive Care Med. 2008 Apr; 34(4): 664â674. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLautrette A, Darmon M, Megarbane B, et al.: A communication strategy and brochure for relatives of patients dying in the ICU. N. Engl. J. Med. 2007 Feb 1; 356(5): 469â478. PubMed Abstract | Publisher Full Text\n\nFine E, Reid MC, Shengelia R, et al.: Directly observed patient-physician discussions in palliative and end-of-life care: A systematic review of the literature. J. Palliat. Med. 2010 May 1; 13(5): 595â603. 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Publisher Full Text\n\nKodali S, Stametz RA, Bengier AC, et al.: Family experience with intensive care unit care: association of self-reported family conferences and family satisfaction. J. Crit. Care. 2014 Aug 1; 29(4): 641â644. Publisher Full Text\n\nGries CJ, Curtis JR, Wall RJ, et al.: Family member satisfaction with end-of-life decision making in the ICU. Chest. 2008 Mar 1; 133(3): 704â712. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarvey MA: Evidence-based approach to family care in the intensive care unit: Why canât we just be decent? Crit. Care Med. 2004 Sep 1; 32(9): 1975â1976. Publisher Full Text\n\nBreu C, Dracup K: Helping the spouses of critically ill patients. Am. J. Nurs. 1978 Jan 1; 78(1): 50â53. PubMed Abstract | Publisher Full Text\n\nWasser T, Pasquale MA, Matchett SC, et al.: Establishing reliability and validity of the critical care family satisfaction survey. Crit. Care Med. 2001 Jan 1; 29(1): 192â196. PubMed Abstract | Publisher Full Text\n\nKryworuchko J, Heyland DK: Using family satisfaction data to improve the processes of care in ICU. Intensive Care Med. 2009 Dec; 35(12): 2015â2017. Publisher Full Text\n\nNorton SA, Hogan LA, Holloway RG, et al.: Proactive palliative care in the medical intensive care unit: effects on length of stay for selected high-risk patients. Crit. Care Med. 2007 Jun 1; 35(6): 1530â1535. PubMed Abstract | Publisher Full Text\n\nLilly CM, De Meo DL, Sonna LA, et al.: An intensive communication intervention for the critically ill. Am. J. Med. 2000 Oct 15; 109(6): 469â475. Publisher Full Text\n\nLautrette A, Darmon M, Megarbane B, et al.: A communication strategy and brochure for relatives of patients dying in the ICU. N. Engl. J. Med. 2007 Feb 1; 356(5): 469â478. PubMed Abstract | Publisher Full Text\n\nSchaefer KG, Block SD: Physician communication with families in the ICU: evidence-based strategies for improvement. Curr. Opin. Crit. 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PubMed Abstract | Publisher Full Text\n\nVenkataraman R, Ranganathan L, Rajnibala V, et al.: Critical care: Are we customer friendly? Indian J. Crit. Care Med. 2015; 19: 507â512. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAzoulay E, Chevret S, Leleu G, et al.: Half the families of intensive care unit patients experience inadequate communication with physicians. Crit. Care Med. 2000 Aug 1; 28(8): 3044â3049. PubMed Abstract | Publisher Full Text\n\nCurtis JR, Burt RA: Point: the ethics of unilateral âdo not resuscitateâ orders: the role of âinformed assentâ. Chest. 2007 Sep 1; 132(3): 748â751. PubMed Abstract | Publisher Full Text\n\nMann HB, Whitney DR: On a test of whether one of two random variables is stochastically larger than the other. Ann. Math. Stat. 1947 Mar 1; 18: 50â60. Publisher Full Text\n\nDodge Y: Kruskal-Wallis test. The Concise Encyclopedia of Statistics.2008; pp. 288â290.\n\nKirch W: Pearsonâs correlation coefficient. Encyclopedia of Public Health. Dordrecht, The Netherlands: Springer; 2008; pp. 1090â1091.\n\nKevin M, Sullivan AGD: Toolkit Shell for developing new applications, OpenEpi. no date. (Accessed: December 7, 2020). Reference Source\n\nMin J, Kim Y, Lee JK, et al.: Survey of family satisfaction with intensive care units: a prospective multicenter study. Medicine. 2018 Aug; 97(32): e11809. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRothen HU, Stricker K, Einfalt J, et al.: Variability in outcome and resource use in intensive care units. Intensive Care Med. 2007 Aug; 33(8): 1329â1336. Publisher Full Text\n\nvan den Broek JM , Brunsveld-Reinders AH, Zedlitz AM, et al.: Questionnaires on family satisfaction in the adult ICU: a systematic review including psychometric properties. Crit. Care Med. 2015 Aug 1; 43(8): 1731â1744. Publisher Full Text\n\nKarlsson C, Berggren I: Dignified end-of-life care in the patientsâ own homes. Nurs. Ethics. 2011 May; 18(3): 374â385. Publisher Full Text\n\nWall RJ, Engelberg RA, Downey L, et al.: Refinement, scoring, and validation of the Family Satisfaction in the Intensive Care Unit (FS-ICU) survey. Crit. Care Med. 2007 Jan 1; 35(1): 271â279. PubMed Abstract | Publisher Full Text\n\nFerrando P, Gould DW, Walmsley E, et al.: Family satisfaction with critical care in the UK: a multicentre cohort study. BMJ Open. 2019 Aug 1; 9(8): e028956. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwarzkopf D, Behrend S, Skupin H, et al.: Family satisfaction in the intensive care unit: a quantitative and qualitative analysis. Intensive Care Med. 2013 Jun; 39(6): 1071â1079. PubMed Abstract | Publisher Full Text\n\nLam SM, So HM, Fok SK, et al.: Intensive care unit family satisfaction survey. Hong Kong Med. J. 2015 Oct 1; 21(5): 435â443. Publisher Full Text\n\nSchaefer KG, Block SD: Physician communication with families in the ICU: evidence-based strategies for improvement. Curr. Opin. Crit. Care. 2009 Dec 1; 15(6): 569â577. PubMed Abstract | Publisher Full Text\n\nKulkarni HS, Kulkarni KR, Mallampalli A, et al.: Comparison of anxiety, depression, and post-traumatic stress symptoms in relatives of ICU patients in an American and an Indian public hospital. Indian J. Crit. Care Med. 2011 Jul; 15(3): 147â156. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamed KA, Buzaid F, AlHafi M, et al.: Assessment of family satisfaction in the intensive care environment: opportunities for improvement. figshare. Preprint. 2022. Publisher Full Text"
}
|
[
{
"id": "167694",
"date": "11 Apr 2023",
"name": "Anis Chaari",
"expertise": [
"Reviewer Expertise Critical Care."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would like to thank you for giving me the opportunity to review the manuscript entitled âA cross-sectional study assessing family satisfaction in the intensive care environment in Bahrain: opportunities for improvementâ.\nThe manuscript is well written with a fluent progression of the ideas. It is dealing with an interesting topic, that is relevant to the quality of care (family satisfaction). Moreover, it is one of the rare studies approaching this aspect in the middle eastern adult intensive care units.\nMajor comments:\nI would advise the authors to review the statistics in table 3. In fact, the first 6 parameters (age, sex, mortality, type of admission, comorbidities, ventilator) are labelled as patients demographics but the total number of each raw is different than 77 which is the total number of included patients. For instance, the age was divided in three categories but the total number (N) of these categories is 94 (different than 77). Similarly, the total number of male and female is 100 which is different than 77 (could it be relatives characteristics rather than patientsâ characteristics?). The same applies for the other parameters included in the table 3.\nMinor comments:\nI would suggest adding more details regarding the unit characteristics (bed capacity, average yearly census) and more details regarding the rules for relatives (visiting time, any privileges given to the relatives of dying patientsâŠ).\n\nThe authors mentioned the number of included patients (77 patients and 100 relatives) in both methods and result sections. This statement can be removed from the methods.\n\nI would suggest to change the title of table 1 to : Relatives evaluation regarding the overall level of ICU care and decision making.\nSimilarly, I would suggest changing the title of table 2 to: Relatives involvement in the decision making. In fact this will avoid the redundancy of the same title in both tables.\nIn summary, I highly recommend Approving the manuscript, provided the above-mentioned revision recommendations are considered.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "252797",
"date": "23 Apr 2024",
"name": "Frank Kiwanuka",
"expertise": [
"Reviewer Expertise AI in Health Care",
"Intensive Care",
"Global Health",
"Machine Learning"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting paper that assessed the needs of patientÂŽs family members in an intensive care unit and identified areas of improvement in quality care. The authors have carefully conducted the study however, there are major issues that need to be addressed to improve the manuscript.\n\n1. Abstract: - At the end of the first sentence the authors mention that \".....and levels of satisfaction have become a measure of intensive care.\" In its current form, the sentence is vague. I suggest that they can change the sentence to e.g., ......and levels of satisfaction have become a measure of quality of care in intensive care units\" - The name of the questionnaire used to evaluate family satisfaction (FS-ICU-24) is wrongly presented. The authors need to correct this to - Family Satisfaction with Care in the Intensive Care Unit - Version 24: (FS-ICU-24). It could also be important to report the language version of the questionnaire e.g., Arabic. - It seems that the reported overall level of satisfaction of 5.04 ± 1.104 is the mean and its standard deviation. First, although there is no recommended format for reporting the mean and the standard deviation, it can be better to clarify that \"the mean level of satisfaction was 5.04 (S.D = 1.04) on a scale of 1-6.\n\n2. Introduction: - The abbreviation 'ICU' is used extensively throughout the paper to refer to Intensive Care Units. It would be helpful to provide the full meaning of all abbreviations used in the main body of the paper when introducing the abbreviations for the first time to ensure clarity for readers who may not be familiar with the term. - The introduction is well written however, most of the references used in this section are very old. There is substantial current literature on the topic. e.g. Padilla Fortunatti C, et.al., 2022 (Ref 1) Peterson MJ, et al., 2022 (Ref 2)\n3. Methods - FS-ICU-24 is mentioned for the first time in the main body of the paper, this needs to be presented in full for the first time in the main body of the paper. - Ethics: Was it voluntary to participate in the study? I suggest that a sentence on the nature of participation (voluntary or otherwise) be added in the ethics subsection. - The authors mentioned the \"......were distributed by KH..\" I assume this is one of the authors but this might be unclear to the readers who/what KH is. Please make the statement clear. - What was the nature of recruitment of participants into the study e.g., was it purposive sampling or? - what were the modifications made to the FS-ICU-24? it could be good to highlight some of them. - In the instrument and data collection section, you mention that you used the web-based FS-ICU-24 questionnaire, did you collect data via an online platform? â or via hard copies distributed to the participants? This is not clear. - Some details about the language of the FS-ICU-24 is missing. - In the statistics part, it is important to include details on how the level of family satisfaction was presented considering that this was measured using the FS-ICU-24.\nResults: -  Without a description of how the authors computed the overall level of satisfaction in the methods section, it makes it difficult to understand how they concluded that \"the overall level of family satisfaction was 5.04±1.104. How did the scale of 1-6 illustrated in Figure 1 computed?\n\nDiscussion: - In most parts of the discussion, the authors have not provided a concrete interpretation of the results and their implications. Although much of the discussion situated the findings within the broader context of existing literature, there is a need to highlight similarities, differences, or contradictions. Discuss how your findings contribute to maintaining or advancing the quality of care in ICUs in your setting and beyond. - Overall, the paper will benefit from professional language editing to improve its readability.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-325
|
https://f1000research.com/articles/12-323/v1
|
23 Mar 23
|
{
"type": "Research Article",
"title": "Effect of facemask, handwashing, and social distancing on contracting COVID-19 infection in Saudi Arabia: a case-control study",
"authors": [
"Fahad M Alswaidi",
"Abdullah M Assiri",
"Muhra M Alalway",
"Haya H Alhaqbani",
"Haya H Alkahtani",
"Abdullah M Assiri",
"Muhra M Alalway",
"Haya H Alhaqbani",
"Haya H Alkahtani"
],
"abstract": "Background: This study aimed to evaluate the impact of self-preventive measures (handwashing, mask-wearing, and social distancing) on preventing the Coronavirus Disease-2019 (COVID-19) infection in Saudi Arabia (KSA). Methods: An unmatched case-control study (1 to 1 ratio) was conducted with a sample size of 1102. The researchers used descriptive, bivariate, and multivariate analyses to determine the effect of handwashing, mask-wearing behavior, and social gatherings on COVID-19 infection. Results: The results evinced that participants who believed that the facemask was important for preventing COVID-19 infection were more likely protected against the disease (OR=0.450; 0.320â0.631). Using a facemask during social visits and shopping indicated a lower protective effect than using it at work (OR=1.299; 1.011â1.668). The type of mask (disposable versus cloth; OR=0.929; 0.590â0.1.462) and its wearing period (1< dayversus â€1 day; OR=0.907; 0.662â1.243) showed an almost neutral statistical association with acquiring the infection. Contrarily, handwashing time for â¥20 seconds, compared to a shorter time, had a statistically significant protective association with the infection even after adjusting the odds ratio (OR=0.616; 0.4763â0.796). It was also noted that gathering with relatives and friends â¥4 times per month exhibited a higher and statistically significant association with the infection compared to the lower times (OR=1.347; 1.046â1.734). Furthermore, people who prayed five times a day at the mosque showed a higher risk of infection than those who prayed less than five times (adjusted OR=1.740; 1.152â2.626). Conclusions: This study suggested that handwashing for more than 20 seconds is the most important preventive factor among all considerations investigated. Moreover, it was noticed that disposable and cloth facemasks may have the same effect regardless of the wearing period or sterilization status. Furthermore, gathering with relatives and friends, as well as praying five times a day at the mosque, were ascertained as significant social factors in acquiring the COVID-19 infection.",
"keywords": [
"COVID-19 in Saudi Arabia",
"Facemask",
"Handwashing",
"Social distancing"
],
"content": "Introduction\n\nInfection control strategies include blocking any stage of the infection pathway. In the case of airborne transmission, this means reducing spread of the pathogen from an infectious person, using disinfection techniques to kill pathogens released, or simply isolating infectious people in special rooms.1 Infection controls generally constitute three categories: administrative, personal protection, and environmental. Administrative controls aim to keep infectious people away from vulnerable people while also ensuring that technical controls, such as personal protection, are used properly.2 For the transmission pathway, personal protection usually involves masks to forestall pathogen distribution or inhalation. Environmental controls primarily intervene after pathogen leaves one person's breathing zone before entering the breathing zone of another.2 The effectiveness of protective measures leans on the strength of surface disinfectants, hand sanitizers, and the materials used to make personal protective equipment. Adequate screening for some infectious diseases, which may be asymptomatic, can play an important role in controlling the transmission.2\n\nQuarantine and isolation are critical public health strategies that frequently necessitate the use of the law to enforce.3,4 Isolation of symptomatic individuals until they are no longer infectious is a common approach to combat viral illnesses, and has the potential to effectively interrupt the transmission and halt an outbreak.5 For instance, policies within healthcare organizations require that healthcare workers who contract influenza are not allowed to return to work until their symptoms have resolved. Another important public health strategy to cope with infections transmitted from person to person involves identifying contacts of infected individuals and providing them with either treatment or prophylaxis. Contact tracing is a crucial element of public health prevention approaches.4\n\nThe study of disease surveillance for the recognition and evaluation of disease patterns can provide information on the danger and magnitude of disease burden to individuals, people, institutions, subgroups of populations, and, therefore, the community at large.6 Effective communication of disease burden and the outcomes achievable through well-managed and effective control programs can be a powerful advocacy tool.2 The challenge facing infectious disease control programs is to design an optimal set of interventions at the local, institutional, community, national, and international levels supported and accepted by the political leadership and the people to whom these measures are applied.7\n\nWhen the COVID-19 pandemic broke out in 2020, without an effective treatment or vaccine and with new virus variants emerging, preventive measures were the only option to restrict its spread.7 Limiting the spread of COVID-19 necessitates the development of coordination mechanisms not only in health sectors but also in other departments, such as transportation, travel, commerce, finance, security, and others.8\n\nPreventive measures are centered mainly on three practices that require individuals' commitment and willingness besides public enforcement. These personal protective measures include handwashing and facemask usage, social distancing, and quarantine.9 Since the beginning of the pandemic, Saudi Arabia implemented the most stringent preventive measures possible, which resulted in the reduction in the number of cases and deaths.10 The preventive measures began in Saudi Arabia on the February 2, 2020 when the government evacuated Saudi students from Wuhan city. Soon after, Saudi airlines suspended flights to China.6 Within weeks of the first confirmed cases, the ministry of health quarantined all visitors entering the country, instituted a contact tracing regimen, closed schools, suspended workplace attendance, prohibited prayers in mosques, and limited pilgrimage to only 50 thousand locals.11 On May 7, 2020, the Ministry of the Interior approved regulations that restricted public gatherings and mandated temperature monitoring, hand sanitizer use, facemask use, and maintenance of a 2-meter distance at public places. Strict penalties against violators of these preventive measures are being imposed to date in some places.12\n\nThis study was initiated to evaluate the impact of self-preventive measures (handwashing, mask-wearing, and social distancing) in preventing the disease. The main aim was to provide a comparative analysis of these interventions and identify the most effective ones. This research will aid public health professionals in designing effective prevention and control policies.\n\n\n\nâ To determine the associations between preventive measures (facemask, hand washing, and social distancing) and contracting COVID-19 infection in Saudi Arabia.\n\nâ To identify and recommend the most effective COVID-19 prevention practices in Saudi Arabia.\n\n\nMethods\n\nEthical approval was obtained from the Ministry of Health (MOH) Instituional Review Board (approval number: 20-213M).\n\nAn unmatched retrospective caseâcontrol study. Administrative proposal approval from MOH was obtained on 01/11/2020 while the remaining study steps took six months until the final report was formulated on 28/4/2021 (Extended data 1).\n\nPotential participants were selected from the Health Electronic Surveillance System (HESN) database of all COVID-19 tested individuals with confirmed results (positive or negative) in October 2020 (15,215 cases). This is the most recent data available to minimize recall bias.\n\nCase: Any confirmed COVID-19 case in the dataset with a positive PCR result and a phone number.\n\nControl: Any case in the dataset with a confirmed negative PCR test result and an available phone number.\n\nAfter downloading the COVID-19 dataset for the assigned period, cleaning was performed to remove duplicates, outliers, and artifacts. Simple random sampling technique was used to select the samples. The required sample size was 386 under the assumptions of a 95% confidence level, alpha value of 0.05, and a COVID-19 infection proportion of 50%. However, since sufficient data were available and a low response rate was expected, we selected 6000 potential participants at random using online random number generator.13 Two frames for cases (2753) and controls (3247) were constructed containing names and phone numbers of the potential participants. After obtaining the appropriate IRB (20-213M) from the Ministry of Health (MOH), the questionnaire in Google forms was sent to all potential participants via the SMS services of MOH. With a response rate of 18.3%, a total of 1102 (551 cases and 551 controls) participants responded to the questionnaire completely. The case to control ratio was 1:1 (Extended data 2).\n\nBefore commencing the questionnaire, participants' consent was sought and documented on the answer form. The questionnaire consisted of five main parts: confirmation of result, i.e., (positive/case) or (negative/control), demographic data, facemask factors, handwashing factors, and social distancing factors (Extended data 3).\n\nData were managed and edited using Microsoft® Excel® (Microsoft® Office 2016, Microsoft® Corp USA). (Microsoft Excel, RRID:SCR_016137) Statistical analyses were performed using Statistical Package for the social Sciences (IBM SPSS statistics 25 USA) (SPSS, RRID:SCR_002865).\n\nCentral tendency and dispersion measures were calculated for the variables, and findings were presented in suitable descriptive tables according to the study objectives. Categorical data of independent variables were compared and tested against the identified dependent variable (outcome) using univariate analysis to obtain the odds ratio as a measure of association. As the final step in the data analysis, multivariate analysis using a binary logistic regression model was implemented to adjust for possible cofounders.\n\n\nResults\n\nThe â€40 years old age group represented 59.3% of all respondents, with a mean age of 37.7±12.6. Males accounted for 61.3%, Saudis for 63.6%, and those from the Central and Western regions for 40.6 and 21.5%, respectively (Table 1).\n\nThe data made it evident that the respondents younger than 40 years of age (OR=1.503; 1.181â1.915) and females (OR=2.072; 1.618â2.653) had a higher and statistically significant chance of contracting COVID-19 than others. Moreover, unemployment (OR=2.485; 1.923â3.213), monthly income less than SAR 6000 (OR=1.482; 1.169â1.880), and a low level of education (OR=1.462; 1.135â1.883) showed significant statistical associations with infection. In addition, living alone reduced the risk of infection by 64% than shared housing (OR=0.362; 0.274â0.479), whereas living with the family doubled the risk of infection compared to living with friends (OR=2.497; 1.451â4.296).\n\nThe respondents who said that the facemask is essential were more likely protected against the COVID-19 infection by nearly 55% (OR=0.450; 0.320â0.631; Table 2). Using the facemask during social visits and shopping indicated a lower protective effect than using it at work (OR=1.299; 1.011â1.668). There was no statistically significant difference between the effect of disposable versus cloth facemasks (OR=0.929; 0.590â0.1.462). Further, facemask wearing periods showed a negligible statistical difference (OR=0.907; 0.662â1.243).\n\nThe participants who believed that handwashing is important were 55% more likely to be protected against the infection (OR=0.451; 0.231â0.881). Handwashing frequency (three times per day) and the use of sanitizers showed minimal protective associations with the infection, but neither was statistically significant. In addition, handwashing for â¥20 seconds evinced a protective statistical association with the disease by 39% (OR=0.616; 0.4763â0.796), compared to the shorter handwashing time.\n\nRespondents who believed that social distancing is important in combating the infection were 59% more likely to be protected than others (Table 3; OR=0.413; 0.241â0.709). Going to work or school (OR=0.353; 0.257â0.452), shopping four times a month (OR=0.506; 0.398â0.642), and visiting cafés and restaurants all had a statistically significant protective effect. Likewise, keeping a distance of â€2 meters, shopping time for more than an hour, and spending more than an hour in a café or restaurant all showed no significant associations with the infection. Moreover, going to the mosque also did not have a statistically significant association with infection; however, the participants who prayed all five prayers per day at the mosque were at a higher risk of infection than those who prayed less than five times (OR=1.740; 1.152â2.626).\n\nSocial factors appeared to play a crucial role in spreading the COVID-19 infection. Our data depicted that gathering with relatives and friends (â¥4 times a month) exhibited a significant statistical association with the chances of contracting the infection (OR=1.347; 1.046â1.734). Similarly, it was also noticed that the entrants who attended social occasions in the previous month were more likely to get infected compared to those who did not (OR=1.848; 1.418â2.407). Nevertheless, the number of events attended and the number of attendees had no significant statistical association with the infection.\n\nBeing informed about COVID-19 also seemed to be a major role player in contracting the virus. The incidence of infection showed a statistically significant protective association with awareness of COVID-19 health messages in the media (OR=0.612; 0.402â0.932). However, those who said that social media was their source of information were at a relatively higher risk of infection (OR=1.668; 1.259â2.208).\n\nOut of all independent variables tested in the bivariate analysis (single variable association), 14 showed statistically significant associations with the outcome variable (positive versus negative). In the final step of the regression model, only eight variables exhibited significant statistical associations (Table 4). Briefly, praying at the mosque five times a day demonstrated a strong and significant statistical association with the chances of infection. While, on the other hand, the participants believing in the importance of handwashing and handwashing for more than 20 seconds indicated a substantial and statistically significant protective effect against the infection.\n\n\nDiscussion\n\nThis study was conducted to assess the effectiveness of self-preventive measures in reducing the risk of COVID-19 infection in the Saudi population. Since 2020, the COVID-19 pandemic has changed the face of the world and brought about significant changes to the lifestyle of people globally. Apart from governmental efforts to address this challenge at the policy level, self-preventive measures are also critical to preventing infection. Against this backdrop, the current study investigated the influence of handwashing, mask-wearing, and social distancing. The main findings support some other similar studies conducted in different parts of the world.\n\nIn this study, younger respondents were found to be at a higher risk of contracting COVID-19 infection. This finding could be attributed to greater social activities among the young population, as documented in earlier COVID-19 studies from Saudi Arabia.14,15 Studies from other countries like Italy, Brazil, and the USA also came to the same conclusion.16 Moreover, the female population in our study appeared to be at higher risk of contracting the disease. Despite the COVID-19 gathering prohibition rules, females in the Saudi community tend to congregate in large numbers in their homes on a regular basis. Hence, our results hinted at a potential role of this behavior in spreading the infection compared to the male population, a finding that contradicted the previous reports from Saudi Arabia.14,15 However, the same finding was reported in seven European countries and the United States.17\n\nThe nationality of the respondents was also identified as an important demographic factor relevant to this study. The rate of infection was higher among the Saudi population than among non-Saudis. In general, gathering in large groups during social occasions is more common in Saudi culture. This social aspect may have increased the risk of infection among Saudis. Further, respondents with lower socioeconomic status were found to be at a higher risk of infection, which is concurrent with previous local and international studies.14,15,18â21\n\nA positive attitude toward wearing a facemask is an important preventive factor. Despite the dispute on the effectiveness of facemasks at the beginning of the pandemic, their use turned to be a significant preventive factor in combating the infection worldwide.22 The type of facemask (disposable versus cloth) showed no difference in the protective effect. A systematic review by Chu et al.,23 investigated the use of facemasks during the pandemic and illustrated that both the surgical/disposable and cloth types provide the same level of protection. In addition, using a disposable or cloth facemask for more than one day without sterilizing or changing it showed no protective difference compared with using it for shorter periods.\n\nMany countries mandate universal masking, especially for healthcare workers. The Center for Disease Control (CDC) recommended the public to wear masks, particularly in crowded areas.7,24,25 Nonetheless, personal protective equipment is in short supply around the world, the efficacy of various types of masks, such as N95 respirators, surgical masks, and cloth masks, is still under investigation, and recommendations on the use, reuse, and sterilization of masks are inadequate.24,25 One of the arguments regarding the facemask usage is that improper use of the mask increases the risk of COVID-19 infection by providing a false sense of protection.7 On the other hand, several randomized controlled trials conducted to examine the evidence for the community's use of masks and respirators suggested protection from the virus and the likely benefits of masks in this context.7\n\nDue to strict preventive measures in work settings compared to social events and shopping places, the work environments seem to be safer regarding COVID-19 infection. Facemask use and compliance with other preventive strategies are generally taken more seriously by workplace managements. As a result, increased use of facemasks may help to slow the spread of the disease. There have been disparities in the usage of facemasks and their effectiveness against the pandemic;24,25 however, this study supports the notion that using any type of proper facemask is effective in preventing the spread of infection regardless of its sterilization status and the period it is worn for.22\n\nSelf-hygiene is critical to one's overall health. Handwashing has consistently been shown to be a critical factor in preventing the spread of communicable diseases. During a pandemic, the most crucial strategy for the population is to wash their hands and frequently use hand sanitizer while avoiding contact with their face and mouth after touching potentially contaminated surfaces7. This study confirms that people who have a positive attitude toward the importance of handwashing have a lower risk of contracting COVID-19. Handwashing for longer periods (â¥20 seconds) appeared to be the most effective method of combating COVID-19 infection, among other investigated preventive measures. Handwashing for an extended period is inconvenient, but it is documented as the best way to sterilize the hand surface.7,8 In this study, the frequency of handwashing per day and the type of sterilizer or soap used compared to only water had no effect on the infection acquisition. This finding contradicts previous research that suggests that using alcohol-based sanitizer or handwashing with soap has a positive effect on reducing the transmission of respiratory infections.7,8\n\nMaintaining safe distance in public places is a critical precaution as COVID-19 spreads via respiratory droplets. Several countries have implemented this strategy since the beginning of the pandemic to reduce the contact rate in the population and the potential contribution of asymptomatic and pre-symptomatic people to disease transmission.7 This study suggests that residents of Saudi Arabia are aware of the importance of keeping a safe distance as a precautionary measure. The distance itself (â¥2 m versus <2 m) made no difference in acquiring the infection. This finding could be attributed to strict adherence to other preventive measures like wearing a facemask and handwashing.\n\nInterestingly, the results of this study hinted that going to school or work during the pandemic had a significant protective effect against infection. Adequate preventive measures are probably better practiced at work and school than at home; however, this point requires further investigation. Gathering with relatives and friends seems to be an important factor in acquiring the infection. This supports the Saudi MOH's claims that most of the community-acquired COVID-19 cases were from family gatherings.26 Therefore, strict penalties were imposed on those who gather in a group of more than 20 persons.12 Despite the negative impact of social distance on mental health, many countries, including KSA, enforced it to reduce disease transmission and protect the vulnerable population.27 Mitigating these mental health effects necessitates daily routines that include a healthy lifestyle, hobbies, virtual social interactions, and mindfulness.27\n\nRoutine social activities like shopping and visits to cafés and restaurants were supposed to be associated with a higher risk of acquiring the infection; however, the results of this study showed that they have preventive effects, probably due to strict preventive precautions at these places. The association between the risk of infection and visits to these locations may be highly dependent on adherence to disease prevention measures and designated Standard Operating Procedures (SOPs). Therefore, more research is needed on this subject. Contrarily, praying at the mosque five times a day seemed to contribute significantly to the spread of the infection. Despite strict preventive measures implemented in mosques, MOH reports revealed that mosques are potential sites of spreading the infection.26 During the curfew at the start of the pandemic, all mosques in the Kingdom, including the two holy mosques in Makkah and Madina, were closed for three months for the first time in the Kingdom's history. That was a difficult decision for both the government and the public, but it indicates commitment and seriousness of the health authority in enforcing preventive precautions against the current pandemic.\n\nOur results evinced that COVID-19 educational messages in the media played an important role in prevention efforts. However, social media appeared to play a misleading role in this context. Because of a lack of checks and users' differing opinions, social media is a source of false and incorrect data about COVID-19. The false messages and conspiracy theories were well received by the general public, posing a significant challenge to the health authorities in KSA as well as around the world.28\n\n\nConclusion\n\nSaudi residents seem to be aware of the significance of precautionary measures in combating COVID-19 spread. This study suggests that handwashing for more than 20 seconds is the most important preventive factor among all factors investigated. Regarding the use of facemasks, it was noticed that disposable and cloth facemasks may have the same preventive effect regardless of the wearing period or their sterilization status. Moreover, common social activities like going to work or school, frequent shopping, and visits to cafés and restaurants showed preventive effects when compared to the homestay, which could be attributed to the strict preventive precautions at these places. However, gathering with relatives and friends and praying five times a day at the mosque were recognized as important factors in acquiring the COVID-19 infection.\n\nA worth mentioning limitation of this study is that it lacks questions on whether the cases or contacts were subject to isolation or quarantine as an important preventive measure.",
"appendix": "Data availability\n\nFigshare: Unmatched case-control dataset (precautionary practices & Covid19 infection) (1).sav, https://doi.org/10.6084/m9.figshare.22258849.v1. 29\n\nThe project contains the following underlying data:\n\n⢠Unmatched case-control dataset (precautionary practices & Covid19 infection) (1).sav\n\nFigshare: Study timeline.pdf, https://doi.org/10.6084/m9.figshare.21952853.v2. 30\n\nThis project contains the following extended data:\n\n⢠Study timeline.pdf\n\nFigshare: Sampling flowchart.pdf, https://doi.org/10.6084/m9.figshare.21952838.v2. 31\n\nThis project contains the following extended data:\n\n⢠Sampling flowchart.pdf\n\nFigshare: The questionnaire, https://doi.org/10.6084/m9.figshare.21973142.v2. 32\n\nThis project contains the following extended data:\n\n⢠The questionnaire.docx\n\nFigshare: STROBE checklist for âEffect of facemask, handwashing, and social distancing on contracting COVID-19 infection in Saudi Arabia: a case-control studyâ, https://doi.org/10.6084/m9.figshare.21959579.v2. 33\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nWe are thankful to our colleagues in the Health Electronic Surveillance System and the General Directorate for the Electronic Applications in Ministry of Health (MOH) for their support in providing raw data of this study and distributing the questionnaire through the MOH short messages services (SMS).\n\n\nReferences\n\nMorens DM, Folkers GK, Fauci AS: The challenge of emerging and re-emerging infectious diseases. Nature. 2004; 430(6996): 242â249. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEyler AA, Chriqui JF, Moreland-Russell S, et al.: Prevention, policy, and public health. Oxford University Press; 2016. Publisher Full Text\n\nNsubuga P, White ME, Thacker SB, et al.: Public health surveillance: a tool for targeting and monitoring interventions. Disease Control Priorities in Developing Countries. 2nd ed.2006.\n\nDetels R: Epidemiology: the foundation of public health. Oxford textbook of public health. 1st ed.2009; pp. 485â491. Publisher Full Text\n\nDowdle WR: The principles of disease elimination and eradication. Bull. World Health Organ. 1998; 76 Suppl 2(Suppl 2): 22â25. PubMed Abstract | Free Full Text\n\nAlboaneen D, Pranggono B, Alshammari D, et al.: Predicting the epidemiological outbreak of the coronavirus disease 2019 (COVID-19) in Saudi Arabia. Int. J. Environ. Res. Public Health. 2020; 17(12): 4568. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGÃŒner HR, HasanoÄlu Ä°, AktaÅ F: COVID-19: Prevention and control measures in community. Turkish. J. Med. Sci. 2020; 50(SI-1): 571â577. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPradhan D, Biswasroy P, Naik PK, et al.: A review of current interventions for COVID-19 prevention. Arch. Med. Res. 2020; 51(5): 363â374. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNussbaumer-Streit B, Mayr V, Dobrescu AI, et al.: Quarantine alone or in combination with other public health measures to control COVID-19: a rapid review. Cochrane Database Syst. Rev. 4(9). PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlmutairi AF, BaniMustafa A, Alessa YM, et al.: Public trust and compliance with the precautionary measures against COVID-19 employed by authorities in Saudi Arabia. Risk Manag. Healthc. Policy. 2020; 13(753): 753â760. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaudi Press Agency: Interior minister approves regulation to limit gatherings and classifies violations and penalties.2020. (7 May 2020). Reference SourceReference Source\n\nSaudi Press Agency: Ministry of interior approves revising regulations on limiting gatherings, updating violations classification schedule.2020. (30 May 2020). Reference Source\n\nFurey E: Random Number Generator. CalculatorSoup- Online Calculators. Reference SourceReference Source\n\nAlswaidi FM, Assiri AM, Alhaqbani HH, et al.: Characteristics and outcome of COVID-19 cases in Saudi Arabia: review of six-months of data (March-August 2020). Saudi Pharm J. 2021; 29: 682â691. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlsofayan YM, Althunayyan SM, Khan AA, et al.: Clinical characteristics of COVID-19 in Saudi Arabia: A national retrospective study. J. Infect. Public Health. 2020; 13(7): 920â925. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOnder G, Rezza G, Brusaferro S: Case-fatality rate and characteristics of patients dying in relation to COVID-19 in Italy. JAMA. 2020; 323(18): 1775â1776. PubMed Abstract | Publisher Full Text\n\nParker A, Maree G, Götz G, et al.: Women and COVID-19 in Gauteng, South Africa.2020. (31 August 2020). Publisher Full Text Reference Source\n\nBoehmer TK, DeVies J, Caruso E, et al.: Changing age distribution of the COVID-19 pandemicâUnited States, MayâAugust 2020. Morb. Mortal. Wkly Rep. 2020; 69(39): 1404â1409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao W, Zhong Z, Xie X, et al.: Relation between chest CT findings and clinical conditions of coronavirus disease (COVID-19) pneumonia: a multicenter study. Am. J. Roentgenol. 2020; 214(5): 1072â1077. PubMed Abstract | Publisher Full Text\n\nConti P, Younes A: Coronavirus COV-19/SARS-CoV-2 affects women less than men: Clinical response to viral infection. J. Biol. Regul. Homeost. Agents. 2020; 34(2): 339â343. PubMed Abstract | Publisher Full Text\n\nEuropean Centre for Disease Prevention and Control: Coronavirus disease 2019 (COVID-19) in the EU/EEA and the UKâeleventh update: resurgence of cases.2020. Reference Source\n\nBrooks JT, Butler JC: Effectiveness of mask wearing to control community spread of SARS-CoV-2. JAMA. 2021; 325(10): 998â999. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChu DK, Akl EA, Duda S, et al.: Physical distancing, face masks, and eye protection to prevent person-to-person transmission of SARS-CoV-2 and COVID-19: a systematic review and meta-analysis. Lancet. 2020; 395(10242): 1973â1987. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMacIntyre CR, Chughtai AA: A rapid systematic review of the efficacy of face masks and respirators against coronaviruses and other respiratory transmissible viruses for the community, healthcare workers and sick patients. Int. J. Nurs. Stud. 2020; 108: 103629. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTirupathi R, Bharathidasan K, Palabindala V, et al.: Comprehensive review of mask utility and challenges during the COVID-19 pandemic. Infez. Med. 2020; 28(suppl 1): 57â63. PubMed Abstract\n\nMinistry of Health Saudi Arabia: Curve still going up in some KSA regions.2021. Reference Source\n\nTeslya A, Pham TM, Godijk NG, et al.: Impact of self-imposed prevention measures and short-term government-imposed social distancing on mitigating and delaying a COVID-19 epidemic: A modelling study. PLoS Med. 2020; 17(7): e1003166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHammad MA, Alqarni TM: Psychosocial effects of social media on the Saudi society during the Coronavirus Disease 2019 pandemic: A cross-sectional study. PLoS One. 2021; 16(3): e0248811. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlswaidi F, et al.: Unmatched case-control dataset (precautionary practices & Covid19 infection) (1).sav. Dataset. figshare. 2023. Publisher Full Text\n\nAlswaidi F, Assiri AM, et al.: Study timeline.pdf. Dataset. figshare. 2023. Publisher Full Text\n\nAlswaidi F, Assiri AM, et al.: Sampling flowchart.pdf. Dataset. figshare. 2023. Publisher Full Text\n\nAlswaidi F, Assiri AM, et al.: The questionnaire. Dataset. figshare. 2023. Publisher Full Text\n\nAlswaidi F, Alalawi M, et al.: STROBE-case-control checklist 26.01.2023.pdf. Dataset. figshare. 2023. Publisher Full Text"
}
|
[
{
"id": "233152",
"date": "27 May 2024",
"name": "Joyzy Pius Egunjobi",
"expertise": [
"Reviewer Expertise psychology",
"psycho-spiritual therapy",
"neuropsychology",
"marriage and family",
"addictive disorders"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study analyzed the effectiveness of self-preventive measures in reducing COVID-19 infection in Saudis. Demographic factors, such as younger respondents, females, and nationality, were identified as significant contributors to infection risk. Facemasks were reported to have a significant preventive factor, with both disposable and cloth types providing protection. Work environments were reported to be safer due to strict preventive measures. Longer handwashing periods were reported to be the most effective method of combating infection. Social distancing did not significantly affect infection acquisition. School or work were found to be protective, while gathering with relatives and friends was a significant factor. Media education messages were also important in prevention efforts.\nThere is no doubt that these measures have been reported in many studies to prevent the spread of COVID-19 (Chiu et al., 2021[1]; Dearling et al., 2021[2]; Kwon et al., 2021[3]; Egunjobi, 2020[4] , Egunjobi, 2020 [5]) A critical review of the data between those who tested positive and negative revealed that the gap between those who considered facemasks important or not is slightly different (between nearly average [45%] and slightly above average [55%]). This reflects, in part, the researchersâ bias, which may partly affect the significance of the conclusion of the study.\nNotwithstanding my observation, the study is consistent with previous studies that found that facemasks, handwashing, and social distance were truly preventive. So, according to the 1500 Dutch philosopher Desiderius Erasmus, 'prevention is better than cure' (Royal College of Nursing, n.d.- https://www.rcn.org.uk/Get-Involved/Campaign-with-us/Prevention-is-better-than-cure#:~:text=Promotion%20of%20healthy%20lifestyles%20and,Desiderius%20Erasmus%20in%20around%201500.), and prevention costs less than treatment (Egunjobi, 2020[4]).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-323
|
https://f1000research.com/articles/12-321/v1
|
23 Mar 23
|
{
"type": "Genome Note",
"title": "The complete genome sequences of Penicillium concavorugulosum",
"authors": [
"Khosi Ramachela",
"Galaletsang Segone",
"Galaletsang Segone"
],
"abstract": "The fungal genus Penicillium and many other soil-borne fungi have widely been reported to create soil myco-rhizhospheric conditions that influence plant growth. These fungal species are relatively difficult to differentiate to species level. Of the three Penicillium species that were morphologically identified, one isolate was established to have a bio-suppressive effect on Fusarium oxysporum. Molecular identification using the polymerase chain reaction (PCR) technique was carried out to accurately identify this isolate to species level. The BLAST consensus and alignments of related species was carried out. The species was identified as Penicillium concavorugulosum (NCBI accession number MK841454.1).",
"keywords": [
"Genome",
"assembly",
"Penicillium concavorugulosum",
"Polymerase chain reaction (PCR)"
],
"content": "Introduction\n\nPenicillium species are a widespread group of facultative fungi which are found in various habitats such as soil, air and decaying plant matter (Pitt and Hocking, 2009; Visagie et al., 2005). Various studies have reported the genus to contain several species which play different roles in agriculture and human health. For example, species such as Penicillium cyclopium and Penicillium citrinum have been reported to be contaminants in corn, soybean, and dried beans (Munkvold et al., 2019). Whereas Penicillium expansum and many other Penicillium species have been reported to cause post-harvest losses in pome fruits and apples (Pitt and Hocking, 2009; Wu et al., 2019).\n\nMany Penicillium species produce extracellular enzymes such as cellulolytic enzymes, polysaccharases and pectic enzymes that aid in the breakdown of organic materials and use in antibiotic production, cell degeneration, as well as food production (Yoon et al., 2007; Rabha and Jha, 2018). Production of these enzymes and phytohormones by Penicillium fungal species that influence plant physiological processes was also reported by (Chanclud and Morel, 2016). Tiwari et al. (2011) also highlighted Penicillium species as some of many fungal microorganisms that possess multifunctional properties that enable them to be used in various agro-industries, biological, medicinal, and commercial purposes.\n\nThis potential widespread use of different fungal species, including Penicillium species, require accurate identification. The need for accurate identification of various important micro-organisms has led to the development and utilization of molecular identification techniques. This technology uses polymerase chain reaction (PCR). Fungal microorganisms are mostly analyzed using the PCR method to determine the ITS region for accurate differentiation of species amongst the genus (Johnston, 2011).\n\nOne of the three Penicillium fungal species was established to possess multifunctional properties such as antibiosis, competition and myco-parasitism, and therefore was considered valuable for use as a bio-control agent against soil fungal pathogens (Segone, 2021). Furthermore, complete genome sequences for this Penicillium fungal species would also contribute to taxonomic studies, and evolution processes as well as understanding its various inherent antagonistic properties.\n\n\nMethods\n\nFour Penicillium species were isolated by use of soil serial dilution procedure. Soil was collected from different sites at the North-West University Mafikeng agricultural farm (25.8278° S, 25.6079° E). These sites had previously been subjected to different agronomic practices such as minimum soil tillage, weed control, fertilizer management and controlled irrigation.\n\nDNA was extracted from the isolate mycelium by use of the Quick-DNA fungal/bacterial Miniprep Kit (Zymo Research, Catalogue No. D6005) (White et al., 1990).\n\nAmplification of the target genes was carried out by use of OneTaq Quick load 2ÃMaster Mix (NEB, Catalogue No. M0486) with primers presented in Table 1. Each Eppendorf tube comprised 10ÎŒL of NEB OneTaq 2X MasterMix with Standard Buffer (Catalogue No. M0482S), 1ÎŒL of genomic DNA (10-30ng/ÎŒL), 1ÎŒL forward primer (10ÎŒM), 1ÎŒL reverse primer (10ÎŒM), and 7ÎŒL nuclease-free water (Catalogue No. E476). Amplification cycles had an initial denaturation at 94°C for 10 minutes, 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C, elongation at 68°C for 1 minute and final elongation at 72°C for 10 minutes. The PCR amplicons were stored at 4°C until electrophoresis.\n\nThe integrity of the PCR amplicons was established by using a 1% agarose gel (CSL-AG500, Cleaver Scientific Ltd) in which EZ-vision Bluelight DNA Dye was used as a stainer. The extracted fragments were sequenced in the forward and reverse direction (Nimagen, BrilliantDyeTM Terminator Cycle Sequencing Kit v3.1, BRD3-100/1000). These were purified by use of Zymo Research, ZR-96 DNA Sequencing clean-up KitTM, (Catalogue No. D4050). The purified fragments were analysed on the ABI 3500xl Genetic analyser (Applied Biosystems, ThermoFisher Scientific). This was done for each reaction and every sample. CLC Bio Main Workbench v7.6 was used to analyse the ab1 files generated by the ABI 3500xl Genetic analyser and the results were obtained by conducting a BLAST search (NCBI) (Stephen et al., 1997).\n\n\nResults\n\nThe genome assembly for the test Penicillium fungal species yielded a total sequence length of 570 with an N50 value of 796.6 bits (882), Expect = 0E00, Identities = 441/441 (100%), Gaps = 0/441 (0%).\n\nThis Penicillium fungal isolate was identified as Penicillium concavorugulosum (Accession: MK 841454.1).",
"appendix": "Data availability\n\nNCBI GenBank: Penicillium concavorugulosum voucher NWUSeq42 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence. Accession number: MK841454.1; https://identifiers.org/ncbiprotein:MK841454.1 (Ramachela and Segone, 2023)\n\n\nAcknowledgements\n\nThe authors would like to thank Northwest University Food Security and Safety Niche Research Entity for funding the Research and Inqaba Biotechnical Industries molecular analysis and sequencing. Part of this work has been published as an MSc. Dissertation by the co-author Miss Galaletsang Segone.\n\n\nReferences\n\nChanclud E, Morel JB: Plant hormones: a fungal point of view. Mol. Plant Pathol. 2016; 17(8): 1289â1297. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnston CL: Identification of Penicillium species in the South African litchi export chain (Doctoral dissertation, University of Pretoria).2011.\n\nMunkvold GP, Arias S, Taschl I, et al.: Mycotoxins in corn: Occurrence, impacts, and management. Corn. AACC International Press; 2019; (pp. 235â287).\n\nPitt JI, Hocking AD: Primary keys and miscellaneous fungi. Fungi and food spoilage. Springer; 2009.\n\nRabha J, Jha DK: Metabolic diversity of penicillium. New and Future Developments in Microbial Biotechnology and Bioengineering. 2018; 217â234. Publisher Full Text\n\nRamachela and Segone: Penicillium concavorugulosum voucher NWUSeq42 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence. [Data set]. GenBank. 2023. Accession number: MK841454.1\n\nSegone GP: Analysis of efficacy of different Penicillium fungal species as bio-control agents against Fusarium oxysporum (Doctoral dissertation, North-West University (South Africa)).2021.\n\nStephen FA, Thomas LM, Alejandro AS, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389â3402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTiwari KL, Jadhav SK, Kumar A: Morphological and molecular study of different Penicillium species. Middle-East J. Sci. Res. 2011; 7(1): 203â210.\n\nVisagie CM, Houbraken J, Frisvad JC, et al.: Identification and nomenclature of the genus Penicillium. Stud. Mycol. 2005; 53(1): 53â62.\n\nWhite TJ, Bruns T, Lee SJWT, et al.: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications. 1990; 18(1): 315â322. Publisher Full Text\n\nWu G, Jurick WM II, Lichtner FJ, et al.: Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay. PeerJ. 2019; 7: e6170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoon JH, Hong SB, Ko SJ, et al.: Detection of extracellular enzyme activity in Penicillium using chromogenic media. Mycobiology. 2007; 35(3): 166â169. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "167524",
"date": "26 Apr 2023",
"name": "Anthony A. Adegoke",
"expertise": [
"Reviewer Expertise Medical/Molecular and Public Health Microbiology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript was poorly presented. All that could be deducted from the manuscript was that amplicon-based sequencing was done. It was not clearly presented. It looked like Sanger sequencing which of course contradicts the title of the manuscript (complete sequencing). Only PCR and gel electrophoresis were stated but for the mention of genetic analyzer.\n\nThe bioinformatic analysis of the sequenced data was not clearly enumerated.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? No\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? No\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-321
|
https://f1000research.com/articles/12-319/v1
|
23 Mar 23
|
{
"type": "Research Article",
"title": "Convolutional neural networks for real-time wood plank detection and defect segmentation",
"authors": [
"Mazhar Mohsin",
"Oluwafemi Samson Balogun",
"Keijo Haataja",
"Pekka Toivanen",
"Oluwafemi Samson Balogun",
"Keijo Haataja",
"Pekka Toivanen"
],
"abstract": "Background: Defect detection and segmentation on product surfaces in industry has become one of the most important steps in quality control. There are many sophisticated hardware and software tools used in the industry for this purpose. The need for the real-time classification and detection of defects in industrial quality control has become a crucial requirement. Most algorithms and deep neural network architectures require expensive hardware to perform inference in real-time. This necessitates the design of architectures that are light-weight and suitable for deployment in industrial environments. Methods: In this study, we introduce a novel method for detecting wood planks on a fast-moving conveyor and using a convolutional neural network (CNN) to segment surface defects in real-time. A backbone network is trained with a large-scale image dataset. A dataset of 5000 images is created with proper annotation of wood planks and defects. In addition, a data augmentation technique is employed to enhance the accuracy of the model. Furthermore, we examine both statistical and deep learning-based approaches to identify and separate defects using the latest methods. Results: Our plank detection method achieved an impressive mean average precision of 97% and 96% of global pixel accuracy for defect segmentation. This remarkable performance is made possible by the real-time processing capabilities of our system, which can run at 30 frames per second (FPS) without sacrificing accuracy. Conclusions: The results of our study demonstrate the potential of our method not only in industrial wood processing applications but also in other industries where materials undergo similar processes of defect detection and segmentation. By utilizing our method, these industries can expect to see improved efficiency, accuracy, and overall productivity.",
"keywords": [
"Artificial Intelligence",
"Convolutional Neural Networks",
"Data Augmentation",
"Defect Detection",
"Deep Learning",
"Neural Networks."
],
"content": "Introduction\n\nDefect detection and segmentation are crucial steps for quality control in automated production lines in various industries such as wood, textiles, and medicine.1â3 These processes involve the use of machine-learning algorithms to identify and classify defects, which can be surface imperfections, structural issues, or other abnormalities that affect product quality. Although it is easy for humans to detect and recognize defects on product surfaces, machines are not always accurate at performing this task. Therefore, industries require automatic defect detection systems in their quality control processes, ensuring that only high-quality products are released for sale. These systems take input images and produce segmentation of areas containing defects.4 The location of the defect is crucial in these applications, and real-time processing is important in industrial environments.\n\nThere have been significant developments in deep learning-based defect detection in recent years, with attempts to create generic datasets5 that can be used for all types of defect detection systems. However, every application requires datasets in a specific industry domain, such as wood and metal.1,6 If a model is trained on a specific defect dataset, it may not always produce the same accurate results on other defects datasets as the properties of the product surface may have different background colors or different types of defects.7 Methods8 have been developed that are trained on different datasets and use knowledge transfer to perform defect detection on other datasets; however, generic defect detection does not seems to work well for all types of defects.\n\nOur paper introduces a new approach for the automated detection and segmentation of defects on rapidly moving wood plank surfaces, using our novel method. Our method first detects the wood plank itself and then the extracted plank image is passed to another module, where defect segmentation is performed in real-time. The input frame is divided into two parts, each with regions of interest (ROI). When a plank enters the first ROI and its front is captured, it is assigned an identification number (ID). The machine then flips the plank and it enters into the second ROI to capture the other side using the same ID. Defects are detected and segmented on both sides, the final classification of the plank is determined based on the severity of the detected defects. We classified wood planks into six categories based on their quality, with 1 indicating a higher quality plank and 6 indicating the lowest quality. Table 3 shows the defect types and corresponding severity of the defects. Our approach uses both sides of the wood panel to achieve better results than previous research, which typically used only one side of the wood surface or employed multiple cameras, resulting in increased hardware costs.9,10 Figure 1 provides a visual representation of the industrial environment in which a wood plank is moving on a conveyor, making it easy to understand how our approach is implemented.\n\nOur contribution in this paper is threefold:\n\n⢠We propose a novel CNN-based approach that can accurately detect wood planks in real-time and perform segmentation of any surface defects present on the planks.\n\n⢠We created a dataset of 5000 labeled images for wood defect segmentation.\n\n⢠We trained our novel model, deployed it on an edge device, and optimized it for use in industrial settings.\n\nIn the section âliterature reviewâ, we review the most recent methods for defect detection and segmentation, with a focus on the wood industry. In the section âmethodsâ, we describe our novel method, and in section dataset, we explain the details of our dataset. The training of our model is described in the section âtrainingâ. We evaluate our method in section evaluation. Finally, we conclude the paper and sketch some new future research work ideas in the section âconclusion and future workâ.\n\n\nLiterature review\n\nThe objective of defect detection and segmentation is to automatically identify patterns on product surfaces during quality control. This is an important task in industrial quality control and manufacturing, and numerous methods have been developed to address this issue. Some of the methods used include k-means clustering,11 active contours,12 region growth,13 and graph cuts14 and deep learning techniques, such as CNN,15 encoder-decoder models,16 R-CNNs,17 recurrent neural network models,18 and generative adversarial network models.19 However, most of these approaches are performed on a single image and executed offline. These methods are not sufficiently efficient for real-time industrial applications, where efficiency is a key consideration.\n\nSeveral techniques have been suggested for identifying defects in various materials and surfaces using statistical methods. For example, the method described in20 employed fuzzy connected components to identify defects present on strip steel surfaces by calculating the maximum and sum of fuzzy connected areas, resulting in a detection rate of 96.8%. In,10 an artificial vision-based system for evaluating the quality of slate slabs is presented, using 3D and 2D color data that is processed and analyzed to detect six specific traits. An unsupervised approach to identifying defects in images was proposed in.21 The method focuses on surface texture and utilizes low-rank representation combined with a texture prior. However, the effectiveness of the method is partially contingent on the quality of the prior map and assumes that the defects are in the foreground, implying that if the background is more prominent than the defect, the method may not be able to detect it.22 employs random decision forest methods for defect detection. This method combines feature extraction and classification techniques to detect defects in fabrics. The advantage of using random decision forests is their ability to handle both continuous and discrete variables, prevent overfitting as a classifier, and perform efficiently when handling large datasets. In,23 traditional classification methods such as local binary patterns and gradient local binary patterns were used to classify defects on the surface of birch veneer. However, the proposed method classifies only two types of defects, cracks and mineral lines, and fails to classify other forms of defects, which is a key limitation. To determine the location of defects in an image,24 proposed the use of gradient local binary patterns. This method leverages the non-continuity of pixels within a local area, reducing the potential area of defect existence, improving accuracy, and saving time for further defect detection. The detection of defects on complex pattern surfaces, such as fabrics, was also explored using traditional statistical methods.25\n\nIn addition to traditional methods, deep-learning based techniques have been used to address the problem of defect detection. Many deep learning models were originally trained to detect a variety of objects, such as people, animals, cars, and other objects in real-world scenes.26 These models are typically trained on a large-scale image datasets, such as microsoft common objects in context (MS-COCO)27 and imageNet large scale visual recognition challenge (ILSVRC).28 For defect detection and other industrial quality assurance tasks, these pre-trained models can be re-trained using a technique called transfer learning to adapt them to the target task of defect detection and classification.26 Many recent methods for defect detection and classification in industrial settings use transfer learning and train CNN-based deep learning models, such as ResNet,29 RetinaNet,30 AlexNet,31 DenseNet,32 VGG1633 and GoogleNet34 to detect, classify and segments defects. The result from these show that all CNN based deep learning methods significantly improve the final prediction accuracy of the detection of defects as compared with the traditional methods.\n\nA wood knot detection and classification method based on a residual network, called TL-ResNet34,1 was proposed. The results from the method claims that TL-ResNet34 is far more accurate than other methods for detection and classification. A weakly supervised CNN-based method for detection and classification was proposed in.35 The model was trained using a small number of labeled images. One limitation of this method is underfitting, in which the model fails to detect and classify different types of defects. An automatic visual inspection system9 has been proposed that can be used to detect and classify defects on wood surfaces. The main contribution of this method is speed optimization of the defect identification task. The results showed that data augmentation and transfer learning techniques can be used together to achieve good results. The pre-trained ResNet152 neural network model achieved an average accuracy of 80.6%.\n\nA deep regression and classification-based framework for defect detection was developed in,36 which has four modules: detection, false positive reduction, connected component analysis, and classification. The proposed method has good accuracy, but it is too computationally intensive for even small input images, and thus, it is limited to offline usage. Another method37 combines a neural architecture search and one of the most famous instance segmentation methods, Mask-RCNN,38 for the detection and segmentation of defects on the surfaces of wood veneers. Regarding the detection of defects on wood surfaces, the proposed method is more accurate and faster than other currently available techniques. However, the segmentation task requires significant amount of time, making it unsuitable for real-time industrial inspection systems. Most of the proposed methods only focused on accuracy, and very few methods have improved efficiency. An improved single shot detector39 based method was proposed to improve the detection efficiency. The trained model detected very few types of defects and was limited. A mixed-FCN (fully convolutional neural network) method was proposed in,15 which is an improved, fully convolution network (FCN) for the detection and recognition of wood defects that outperforms the existing methods while requiring little or no image preprocessing for feature extraction. The model was trained to identify only six types of wood defects. A FCN and regional convolutional neural network (R-CNN)-based method40 were proposed to detect and segment building cracks. The proposed method has limitations owing to its low performance in real-time applications, similarly to other detection and segmentation methods. An improved CNN based method for weld classification was proposed in.41 This method uses image convolution to enhance edge features and combines them with integral images to create a more accurate segmentation. The algorithm can extract the weld edge and divide the region quickly and accurately while maintaining the processing time within the real-time requirements.\n\nIn,42 a modification was made to a CNN-based method named U-Net, replacing its softmax layer with a random forest to detect small surface defects with high accuracy. This method is slow and is limited to an offline setup. A CNN based method was proposed in43 to segment defects on standing trees using LIDAR (light detection and ranging) data. The input data for this method are the point cloud data. A mesh is reconstructed from the point cloud data. Then reconstructed mesh is then used to make a relief map and is taken as input to the U-net for segmentation. This method is computationally expensive and suitable only for offline applications. To reduce the computation time of CNN,44 proposed a method that uses a non-subsampled shearlet transform (NSST) to preprocess images. Then, the images were passed to the CNN for detection and classification. This method has the advantage of faster training speed; however, the speed of inference is not conducive to its utilization in industrial applications.\n\nIn addition to CNN-based deep learning methods, auto-encoders and generative adversarial networks have also been used for defect detection. The dual auto-encoder generative adversarial network (GAN) method,45 a deep learning method, was proposed for defect detection in different types of products. The GAN has the benefit of generating a large amount of data that can be used for training, which makes the model more accurate for predicting defects in unseen data.\n\nIn the literature, most researchers have used classical image processing methods or deep learning methods to extract features to detect and classify defect locations. Most of these methods are offline and unsuitable for real-time industrial use. These methods have many limitations, such as inference speed and detection accuracy. In all these methods, the focus is only on detecting and classifying defects on the plank/wood surface, and none have focused on detecting the wood plank itself. Therefore, we propose a novel CNN method that can detect and classify wood planks and then detect, classify, and segment defects on wood plank surfaces, which can be deployed in industrial environments and outperforms all these methods.\n\n\nMethods\n\nThe automatic detection of wood planks and separating it from the background on a fast-moving conveyor in real-time is a challenging task. Furthermore, defect segmentation on the surface of wood planks in real-time adds more to task complexity. We propose a novel method consisting of a backbone network that performs feature extraction, a detection algorithm for wood plank detection, and a segmentation module, that performs defect segmentation in real-time. Finally, each plank is classified into different categories, from level 1 to 6, depending on the severity of the defects on each plank surface. Figure 2 shows the architecture of the proposed method. Details of these networks and modules are described in the following sub-sections.\n\nHigh prediction performance in CNN training requires a substantial amount of annotated datasets, but acquiring such a large quantity of data can be challenging and expensive, especially for image labeling.46 To address this issue, transfer learning is often utilized with a limited number of datasets, demonstrating its effectiveness as a solution. This occurs when the backbone network is first trained with such a large dataset.\n\nThe backbone network, also known as the baseline network, is responsible for extracting features from input images. There are several state-of-the-art deep CNNs, such as VGG,33 GoogleNet,34 AlexNet,31 and ResNet,29 that can be used as feature extractors or backbone networks for object detection. These networks are known for their accuracy, but they may not be as efficient in terms of inference speed.\n\nWe use MobileNetV347 as the backbone network for feature extraction. The network employs depthwise separable convolutions instead of traditional convolutions to reduce computational complexity. Standard convolutions have spatial dimensions and input/output channels and require a significant number of multiplications. By contrast, depthwise separable convolutions splits the standard convolution into two separate operations: a depthwise convolution that applies filters to each input channel and a 1x1 pointwise convolution that combines the outputs from the depthwise step. This approach results in a smaller model size, fewer parameters, and faster computation times.\n\nTo improve the classification accuracy, we pre-trained MobileNetV3 on a large-scale image dataset called MS-COCO.27 The COCO 2019 object detection datasetâs training and validation subsets, comprising of over 200,000 images, were downloaded. The dataset can be accessed for free on the website. The last three layers, in conjunction with the fully connected layers, are then fine-tuned on the wood planks and defect dataset. Figure 3 shows the MobileNetV3 block containing an input, an expansion convolution, depthwise convolutions, projection layer, and output layer. A residual connection is established if the input and output have the same number of channels.\n\nWe use Faster-RCNN48 network with MobileNetV3 backbone to detect wood planks. The detector extracts features from different layers of a pre-trained backbone network and sends them to a regional proposal network (RPN) and region of interest (ROI) pooling module. The RPN is responsible for identifying the location of potential wood planks in the input image, whereas the ROI pooling module extracts fixed-sized window features and passes them to the final two fully connected layers for class and bounding box predictions. The network takes an input image of size HÃW with three color channels. The first layer is a 3x3 convolutional layer with a stride of two, which reduces the spatial dimensions of the image by half and outputs 40 feature maps. Hardswish49 is used as an activation function Equation 1, which is a nonlinear activation function. The subsequent layers are a series of mobile inverted residual bottleneck (MBConv) blocks. Each block consists of a depthwise convolutional layer followed by a pointwise convolutional layer, with skip connections between the input and output of the block. The MBConv2 block has a stride of two, while the rest have a stride of one. The dilation for all the convolutional layers is one, meaning that there is no dilation applied. After the MBConv blocks, a feature pyramid network (FPN) is used to generate feature maps of different spatial resolutions. The FPN is used to address the problem of scale variance in plank detection, where planks of different sizes may require different spatial resolutions for detection. The FPN in this network produces feature maps with a spatial resolution of H16ÃW16 and 256 feature maps. The RPN (region proposal network) generates object proposals at different locations and scales based on the feature maps from the FPN. The RoIAlign layer then extracts features from each proposal and feeds them into two parallel fully connected layers: a classification layer (Cls) that outputs a probability distribution over the classes (including background) for each proposal, and a bounding box regression layer (BBx) that predicts the offsets to the default bounding boxes for each proposal.\n\nThe final output of the network is a set of class probabilities and bounding box offsets for each anchor box, which can be used to generate the final plank detections. The configuration details of the detection module is shown in Table 1.\n\nIn addition to wood plank detection, the process of defect segmentation involves identifying and separating each pixel from the wood plank belonging to a defect. Each defect type is labeled with a specific color. To extract features at multiple scales and to reduce the computational complexity of the model at the inference stage, we use atrous convolution operations and the atrous spatial pyramid pooling (ASPP) module in the segmentation part. Atrous convolution, is a type of convolution operation in which the filter kernel is dilated with zeros before being applied to the input signal. This allows the operation to have a larger receptive field, without increasing the number of parameters. The ASPP module is a type of multi-scale pooling operation that utilizes atrous convolutions at different dilation rates to capture information at multiple scales. It is used for semantic segmentation to improve the networkâs ability to capture objects of varying sizes. The ASPP module consists of parallel atrous convolution branches with different dilation rates, followed by global average pooling, which aggregates the information across all spatial positions. The output of each branch is then concatenated to form the final feature representation.\n\nThe first layer of the network is a 3x3 convolutional layer with stride two, which reduces the size of the image and increases the number of channels. This layer helps to extract features from the input image. The MobileNetV3 blocks are then used to further extract features from the image. These blocks use depthwise convolutions to reduce the number of parameters in the network while still maintaining good performance. Hardswish is used as an activation function Equation 1.49 By using MobileNetV3 blocks, the network is able to learn features that are specifically relevant for identifying defects in images. After backbone, the ASPP module is used to capture information at multiple scales in the image. This module applies 1x1 convolutions with different dilation rates to the feature map in order to capture information at different scales. This helps the network to identify defects of different sizes and shapes. The decoder module is then used to upsample the feature map back to the original resolution of the input image. This module creates a segmentation map that matches the size and shape of the input image. Finally, the logits layer outputs a probability distribution over the classes of interest which are the defect and non-defect regions of the image. The Softmax activation function50 then create a probability distribution that identify the location of defects in the image. Table 2 shows the complete configuration of the segmentation module along with backbone network.\n\n\nDataset\n\nOur dataset consists of labeled images of planks and defects. For a period of one month, a machine vision camera was installed in a wood industry located in Finland to capture images. The planks move very fast on a conveyor; therefore, we used multi-threading and hardware acceleration to process the high frame rates and capture sharp images. A total of 5000 images were collected using this equipment. These images were visually inspected and discarded if they were blurry or if there was no plank at any particular time. The dataset contains six classes of defects (Table 3) for defect segmentation and two classes for plank detection (plank or background). The dataset is distributed among subsets, such as training, testing, and validation subsets (Table 4). To segment the defects accurately and reduce the number of false positives, we labeled the wood planks themselves and created a dataset for the detection method. We then created a dataset for the defect segmentation model, which takes the extracted plank image as input and draws a segmentation mask for each defect. Currently we are unable to share this full dataset therefore, an example dataset has been created so that is possible to test the proposed methods51\n\nWe categorized each plank according to different defect levels. A plank can contain multiple defects of different classes. Each defect is categorized based on different severity level. The final classification of a plank is determined based on the highest severity of the defect. The Figure 4 shows different defect types. 3 shows the defect types, defect severity, and properties of the defects.\n\nWe used the CVAT (Computer Vision Annotation Tool)52 to label the images in our dataset. This open-source tool was deployed on an Amazon Web Services53 virtual machine. The complete dataset was uploaded to storage and then loaded into CVAT. Each frame from the dataset was labeled individually by drawing a rectangle on the frame for the wood plank dataset and a polygon around the defects for the defect segmentation dataset, and assigning a class name. After annotating the training set, the JSON (javascript object notations) annotation labels and images were downloaded for model training. Then dataset is passed through an augmentation process.\n\nTo make the model more generalizable for inference, we used a technique called data augmentation, which included multiple image operations such as random flip, shear, translation, and rotation. The pytorch54 library has builtin functions to perform these operations. After this process, the dataset contained an increased number of images. Table 4 shows the number of images in each subset after data augmentation process. The model is then trained on the training set with increased number of images.\n\n\nTraining\n\nWe used Pytorch54 (RRID:SCR_018536) distributed data-parallel training module to train the model on multiple graphical processing units (GPUs). The model is trained using two NVIDIA Quadro RTX 8000 48GB GPUs. The starting hyperparameter learning rate is set to 0.1, a batch size of 128 images per GPU, a learning decay of 0.01 per 5 epochs and RMSprop as an optimizer at eps=0.0316. We initially configured the number of epochs to 300; however, after observing the loss on the validation set, we implemented early stopping and the training script reduced the number of epochs to 200.\n\nNetwork quantization is a method of reducing the number of bits per weight of the network. Some hardware supports a faster inference speed with a quantized network. We deployed our model on an Nvidia Jetson Xavier device for real-time inference. The device supports quantized models, by default. We tested the model with precision of 8-bit integers (INT8). The number of parameters are reduced by 40% and the inference speed was increased by approximately 3x. The model was fitted to the MS-COCO dataset through a process of quantization aware training by refining a non-quantized version, where activation and weights are quantized to lower precision, without sacrificing the overall accuracy of the model and making it faster for real-time inference. To make the model more efficient, we pruned it to remove redundant elements in the network. This did not affect the accuracy of detection and segmentation in our results.\n\n\nEvaluation\n\nTo evaluate the performance of the model for the detection of planks, we used mean average precision (mAP) metric Equation 2 on the plank test set, and for the segmentation of defects, we used the mean intersection over union (mIOU) and global pixel accuracy on the defects test set. mIOU is used to evaluate the similarity between the predicted segmentation mask and the ground truth segmentation mask, while global pixel accuracy is used to evaluate the overall performance of a semantic segmentation model. It measures the proportion of pixels in an image that are correctly classified by the model. Given a predicted segmentation mask and the ground truth segmentation mask for an image, the global pixel accuracy can be calculated as the ratio of correctly classified pixels to the total number of pixels in the image. The plank detection mAP was 97% and the defects segmentation mIOU was 76%, with a global pixel accuracy of 96%.\n\nThe network takes an input image and utilizes a region proposal network (RPN) to identify possible regions of interest, in this case, region containing wood planks, which are then classified and their bounding box coordinates refined. A classifier and bounding box regressor network are then used to further refine the identified planks and classify them into their respective classes. The output from this process is a set of detected wood planks with their class labels and bounding box coordinates. The resulting bounding box surrounding each plank is then cropped from the output image and passed to the segmentation module. The segmentation module is designed to receive an image containing a wood plank as input, and it specifically focuses on identifying and isolating any defects present on the plank.\n\nAfter processing the input image, the segmentation module produces an image where each defect is highlighted with segmentation and labeled with its respective class color. Using this information, each wood plank is then classified according to the categories listed in Table 3.\n\nFigure 5 shows an input image with two defined ROIs. Figure 6 and Figure 7 show the outputs from the detection and segmentation modules, respectively. The final output after plank classification is shown in Figure 8.\n\nTo evaluate the real-time performance of our trained model, we deployed it in a wood industry where wood planks move rapidly on a conveyor. We used the Nvidia Jetson AGX Xavier as the processing unit for real-time video encoding, model inference, decoding, and producing output to an external monitor or remote sink. For the video capture, we used an Allied Vision camera system with an adjustable lens. The video capture frame rate was tested with variable settings ranging from 15 to 50 fps. The ideal frame rate to capture the fast-moving wood plank was 25-30 FPS to avoid blurriness and capture sharp frames. The model performed very well without any latency issues at 30 fps and did real-time wood plank detection, defect segmentation, and final plank classification.\n\nThe hardware specification for the industry setup are shown in the Table 5.\n\n\nConclusion and future work\n\nIn this study, we proposed a novel method for the automatic detection of wood planks and defect segmentation. Our method employs several techniques to improve the accuracy of results. First, data augmentation was used to increase the number of images in the dataset. This technique involved applying various image operations such as random flip, shear, translation and rotation to each image. Second, we utilized transfer learning to improve the ability of our model to detect planks and segment defects on the plank surfaces. The results showed that these techniques were effective in achieving high accuracy in detection and segmentation tasks. Specifically, the highest mean average precision value of 97 and global pixel accuracy value of 96 were achieved. Moreover, our method demonstrated real-time performance at 30 frames per second, making it suitable for industrial wood processing applications. These results also suggest that our method has the potential for application to other industrially processed materials. As a future work, further research could investigate the transferability of our method to other materials and industries. This requires the creation of new datasets with proper annotation and training of the model on these datasets. Moreover, our approach is specific to detecting and segmenting surface defects. The future work could explore the detection and segmentation of other types of defects such as internal defects. Future research could focus on the robustness and adaptability of our approach to changes in the lighting conditions, camera angles, and other environmental factors. One potential approach could be to incorporate online learning techniques, which would allow the model to adapt to environmental changes over time. Our method relies on a convolutional neural network for detecting and segmenting surface defects. Although this has proven to be effective, there are other deep learning architectures that could be explored in future work. For example, recurrent neural networks can be used to analyze the temporal data from a conveyor belt. Similarly, attention mechanisms can be employed to selectively focus on regions of an image that are more likely to contain defects.",
"appendix": "Data availability\n\nMS COCO dataste T.-Y. Lin et al., âMicrosoft COCO: Common Objects in Context,â CoRR, vol. abs/1405.0312, 2014. This project contains the following source data:\n\n⢠COCO 2019 Object Detection Task.\n\nThe annotations in this dataset along with this website belong to the COCO Consortium and are licensed under a Creative Commons Attribution 4.0 License.\n\nThis project is currently ongoing and a non disclosure agreement (NDA) has been signed. We are currently unable to provide the full underlying data for this article due to the terms of the NDA. We have supplied a shortened example dataset that can be used to confirm the functionality of the methods presented in our article. This example dataset is available in the extended data section of the article. 51 At the moment there are certain legal and contractual obligations that prevent us from providing a restricted route of access to the complete dataset. As the project continues, we will keep the issue of data access in mind and will work with the company to determine if and when we might be able to share the complete dataset in the future.\n\nFigshare: Code and Data. https://doi.org/10.6084/m9.figshare.22189003. 51\n\nThis project contains the following extended data:\n\n- Defects.csv\n\n- Dataset.zip (Example shortened dataset)\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nGao M, Qi D, Hongbo M, et al.: A transfer residual neural network based on resnet-34 for detection of wood knot defects. Forests. 2021; 12(2). 1999-4907. Publisher Full Text\n\nShahrabadi S, Castilla Y, Lopez MAG, et al.: Defect detection in the textile industry using image-based machine learning methods: a brief review.2022; 2224: 012010. 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CoRR, abs/1703.06870. 2017.\n\nDing F, Zhuang Z, Liu Y, et al.: Detecting defects on solid wood panels based on an improved ssd algorithm. Sensors. 2020; 20(18). 1424-8220. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZheng M, Lei Z, Zhang K: Intelligent detection of building cracks based on deep learning. Image Vis. Comput. 2020; 103: 103987. 0262-8856. Publisher Full Text\n\nMiao R, Jiang Z, Zhou Q, et al.: Online inspection of narrow overlap weld quality using two-stage convolution neural network image recognition. Mach. Vis. Appl. Jan 2021; 32(1): 27. 1432-1769. Publisher Full Text\n\nDong X, Taylor CJ, Cootes TF: Small defect detection using convolutional neural network features and random forests.Leal-Taixé L, Roth S, editors. Computer Vision â ECCV 2018 Workshops. Cham: Springer International Publishing; 2019; pages 398â412. 978-3-030-11018-5.\n\nDelconte F, Ngo P, Debled-Rennesson I, et al.: Tree Defect Segmentation using Geometric Features and CNN. Reproducible Research on Pattern Recognition (RRPR). Milan, Italy: 2021; volume LNCS 12636: pages 80â100. Publisher Full Text\n\nYang Y, Zhou X, Liu Y, et al.: Wood defect detection based on depth extreme learning machine. Appl. Sci. 2020; 10(21). 2076-3417. Publisher Full Text\n\nTang T-W, Kuo W-H, Lan J-H, et al.: Anomaly detection neural network with dual auto-encoders gan and its industrial inspection applications. Sensors. 2020; 20(12). 1424-8220. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGao Y, Mosalam KM: Deep transfer learning for image-based structural damage recognition. Comput. Aided Civ. Inf. Eng. 2018; 33(9): 748â768. Publisher Full Text\n\nHoward A, Sandler M, Chu G, et al.: Searching for mobilenetv3. CoRR, abs/1905.02244. 2019.\n\nGirshick RB, Donahue J, Darrell T, et al.: Rich feature hierarchies for accurate object detection and semantic segmentation. CoRR, abs/1311.2524. 2013.\n\nAvenash R, Viswanath P: Semantic segmentation of satellite images using a modified cnn with hard-swish activation function. Proceedings of the 14th International Joint Conference on Computer Vision, Imaging and Computer Graphics Theory and Applications - Volume 4: VISAPP. INSTICC, SciTePress; 2019; pages 413â420. 978-989-758-354-4. Publisher Full Text\n\nBridle J: Training stochastic model recognition algorithms as networks can lead to maximum mutual information estimation of parameters.Touretzky D, editor. Advances in Neural Information Processing Systems. 1989; volume 2. . Morgan-Kaufmann; Reference Source\n\nMazhar M: Code and Data â figshare.com. [data and code]. Reference Source2023.\n\nCVAT.ai Corporation: Computer Vision Annotation Tool (CVAT).9 2022. Reference Source\n\nAmazon web services. http\n\nPaszke A, Gross S, Massa F, et al.: Pytorch: An imperative style, high-performance deep learning library. CoRR, abs/1912.01703. 2019."
}
|
[
{
"id": "177501",
"date": "14 Jun 2023",
"name": "Zhenye Li",
"expertise": [
"Reviewer Expertise Computer vision",
"neural network",
"hyperspectral imaging"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt's encouraging to see research tackling the pertinent issue of real-time defect detection and segmentation in industrial applications. Your work addresses a critical need in the industry and promises substantial improvement in process efficiency and accuracy.\nI appreciate the clear outline of your novel method for detecting wood planks and segmenting surface defects. The use of a large-scale image dataset and data augmentation techniques indicates thorough preparation and diligence in your research methodology. However, it would be better if some more comparisons could be added to the experiment part. And by the way, the code you provided can not be downloaded for 403 forbidden error.\nYour reported results are impressive, particularly a mean average precision of 97% for plank detection and 96% global pixel accuracy for defect segmentation. The real-time processing capability of 30 FPS further underscores the applicability of your system in an industrial environment. However, it would be interesting to see a comparative analysis with other similar lightweight architectures. This would give a better understanding of the true effectiveness of your model.\nWhile your study makes significant contributions to industrial wood processing applications, extending the methodology to other materials or industries could increase the breadth of your work. Including a discussion on potential challenges or adaptations needed for these different applications would make your research more comprehensive.\nOverall, the potential impact of your work in improving efficiency, accuracy, and productivity in the industry is commendable. The research is well-conducted with significant results, and I look forward to seeing further developments in this area.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-319
|
https://f1000research.com/articles/11-1044/v1
|
13 Sep 22
|
{
"type": "Research Article",
"title": "Preservation of heritage buildings in Alexandria, Egypt: an application of heritage digitisation process phases and new documentation methods",
"authors": [
"Adel El Menshawy",
"Walid Omar",
"Sherif El Adawy",
"Adel El Menshawy",
"Walid Omar"
],
"abstract": "Background: Throughout the history of the city, the architecture of Alexandria, Egypt, has been in contact with world cultures, especially those of the Mediterranean sphere. Alexandria is rich with cultural features dating back seven thousand years. In Alexandria, the heritage value of the city has decreased because there is no suitable documentation system for these more recent assets. The development of a new technique for preserving heritage buildings is required. For example, image- based techniques can gather data using photography, panoramic photography, and close-range photogrammetry. In this research, we primarily seek to implement Heritage Digitisation Process Phases (HDPP) and establish new documentation methods in architectural conservation and built-heritage preservation, i.e., Virtual Reality (VR) and Website Heritage Documentation (WHD). Methods: The methodology is designed to preserve and manage cultural heritage using HDPP for the promotion of heritage building preservation in Alexandria. Results: The results show that the application of HDPP has led to the creation of a digital database about the Société ImmobiliÚre building, which was chosen as a case study for this research. Conclusions: Implementation of HDPP and usage of new documentation methods i.e., VR and WHD create a digital path to help strengthen its image and connect the place to users, recreational areas are created to communicate and explore the cityâs architectural history.",
"keywords": [
"Image-based techniques",
"Photography",
"Panoramic photography",
"Close-range photogrammetry",
"Heritage Digitisation Process Phases (HDPP)",
"Virtual Reality (VR)",
"Website Heritage Documentation (WHD)"
],
"content": "Introduction\n\nHeritage digitisation uses digital media to conserve cultural or natural heritage. In other words, it âconsists of unique resources of human knowledge and expression. It embraces cultural, educational, scientific and administrative resources, as well as technical, legal, medical and other kinds of information created digitally, or converted into digital form from existing analogue resourcesâ.1 Moreover, digitisation converts the physical data that pertains to movable or unmovable cultural heritage by using digital technologies to create multidimensional digital archives, which provide architectural data about design theories, building materials, and construction methodologies in different time periods.\n\nVirtual heritage is the result of heritage digitisation. It is focused on recreating tangible cultural heritage by making realistic 3D models depicting them. In other words, it is âthe use of computer- based interactive technologies to record, preserve, or recreate artefacts, sites and actors of historic, artistic, religious, and cultural significance and to deliver the results openly to a global audience in such a way as to provide formative educational experiences through electronic manipulations of time and spaceâ.2 Virtualised objects are object models generated from the physical world that surrounds us and the result is placed in a computerâs memory, but the generation process of virtualised objects is different. Hence, virtualised objects or environments result from a measurement process.3\n\nVirtual Reality (VR) is a technology that makes use of computer generated interactive graphics which give the user the sensation of being in a virtual, that is, computer-generated world.4 Virtual heritage is linked to VR, offering a new generation of academics and architects the possibility to use VR technology and tools to create immersive experiences and solutions. Experts have predicted that VR will have an influence on our lives similar to that of the internet or smartphones, with interesting potential in a wide variety of disciplines, including heritage preservation, architecture, education, entertainment, and social action.5\n\nThe Heritage Digitisation Process Phases (HDPP) changes documented data that can be perused by individuals to a digital read-only format with the assistance of machines. The digital world is the quickest growing and developing world. Cultural heritage institutions, world heritage sites, libraries, galleries, archives, and museums vary in sort and size across the world, however, within the last decade, the majority of academics and architects use digital technology for digitisation. Current advancements in computer networks, multimedia, VR, and artificial intelligence have given a great establishment to the digitisation of cultural heritage information. The acquisition and perception of cultural heritage information are firmly linked with techniques such as 3D laser scanning or photogrammetry.6 HDPP can be shaped by academics and architects; creating a digital database about heritage buildings that depend on the sort of data (textual data, images, and drawings) that allows users to acquire full knowledge about the documented database and digital library, which features heritage building assets, thus also strengthening usersâ attachment to the place.\n\nBuilding Information Modelling (BIM) has evolved significantly in managing and documenting cultural heritage. It can now represent, in a virtual environment, the actual conservation state of the buildings under analysis. However, the virtual reconstruction procedure of historical cultural heritage is difficult because the objects to model consist of components whose heterogeneous, complex, and irregular characteristics and morphologies are not represented in the BIM platform libraries.7 Therefore, it is essential to introduce technical and historical approaches in both the BIM environment and the point clouds, to model the different virtual parametric components and achieve a BIM model for the architectural heritage under analysis.8,9\n\nObtaining the parametric building elements from the 3D point cloud is a time-consuming and error-prone manual process, because no automation or platform processes currently exist that can ensure a direct change from the 3D point cloud to full BIM models.10,11 Therefore, once 3D virtual models have been created, the libraries of parametric elements should be generated under Historic Building Information Modelling (HBIM).\n\nMurphy and McGovern developed HBIM to integrate contemporary technology and the BIM approach in cultural heritage documentation. HBIM was first used in 2009.12 HBIM executes the modelling and documenting of architectural elements according to artistic, historical, and constructive typologies. It is designed to preserve and manage cultural heritage using HDPP, which is a four-phase process for the documentation, representation, rendering, and dissemination of heritage building. In addition, HBIM functions as a plug-in for BIM and is considered to be a unique library of BIM parametric objects built from historical data. Furthermore, HBIM serves as a system for mapping the parametric objects onto a 3D point cloud and image survey data. Generally, the HBIM library is built using manuscripts and historical architectural documentation, laser scanning, photogrammetric techniques, and other data obtained from the physical analysis of a building.13\n\nThe application of HDPP has led to the creation of a digital database about the Société ImmobiliÚre building which was chosen as a case study for this research. The Société ImmobiliÚre building represents the features that were prominent in architecture at the time of the buildingâs construction, which was built when Egypt was a British colony and therefore the buildingâs design was influenced by European styles.\n\n\nResearch problem\n\nThis research addresses a local and national problem in Egypt that has focused on the decrease in heritage value of certain buildings due to the lack of maintenance and ill-conceived remodeling and additions, which have been built without consideration for the buildingsâ original styles. Moreover, there has been no strategy for designating this rich heritage and no suitable system to document these assets. There has also been a shortage in locally authorised officials specialising in photographing, documenting, and using available techniques to capture data about heritage objects, such as 3D laser scanning or photogrammetry.14 One of the major problems is there has been no entity entrusted with keeping this architectural archival documentation or handing it over to the National Organisation for Urban Coordination. Without any methodology to conserve heritage buildings, the city risks further deterioration to its identity and historical value over time. Currently, there is no database to help us better understand design theories through on-site methods that illustrate the historical background, architectural features and styles of a building. Furthermore, documentation methods applied to heritage buildings cannot access the documented asset models and their information.\n\n\nResearch aim\n\nThis research aims to explain the process of documenting heritage buildings by creating a methodology based on digital documentation, architectural conservation, and preservation, in addition to discussing a new documentation technique that provides a digital document about heritage buildings to local architectural heritage organisations. Moreover, this study aims to create a digital database based on video and image survey data, historical drawings and maps of the municipality, plan drawings, and reference books. The digital database helps better understand the relevant design theories through on- site methods, which illustrate the historical background of the heritage buildings and their architectural features and styles. In addition, this research aims to create new documentation methods for heritage buildings that provide access to the documented asset models and their associated information, as well as improve the process of documenting real estate that may be included on the lists of Egyptian Properties Protected from Demolition - Law No. 144 of 2006 and its executive regulations.\n\n\nMethods\n\nThe research focuses on heritage buildings in Alexandria, Egypt, and highlights the Société ImmobiliÚre building as a case study. The methodology is designed to preserve and manage cultural heritage using HDPP for the promotion of heritage building preservation in Alexandria. The methodology of this study used a method for developing and disseminating heritage digitisation and divides it into four phases. The first phase is that of documenting, which was about finding and analysing information and documenting authentic data emanating from the cultural and architectural past. The authors gathered data, through contact with Alex Med of Bibliotheca Alexandrina and Sigma Properties Company, from reference books, including the âConservation and Rehabilitation of Alexandria's City Centerâ and the âPatrimoines partagés en Méditerranéeâ books authored by Dr. Mohamed Awad the founder of âThe Alexandria Preservation Trustâ, map and historic photo of Société ImmobiliÚre building (see Figure 1-A and Figure 1-C), and general information about the Société ImmobiliÚre building (Table 1). All of these data were gathered from open access sources, and available through institutes and their official websites. In addition, the authors acquired a map of the building location from the Cityâs Municipal of Alexandria, Egypt (see Figure 1-B), and did image survey data by photography, panoramic photography, and close-range photogrammetry on-site by using DSLR camera with a professional camera tripod, padcaster tripod dolly wheel, smartphone, and DJI Osmo Mobile 3 Gimbal for smartphone. Representation constituted the next phase; after the data has been gathered, they were digitised as a 3D point cloud by using a photogrammetry platform, RealityCapture platform (see Software availability statement for alternatives). Next, the rendering phase produced 3D virtual models by using the BIM platform, Autodesk Revit platform (see Software availability statement for alternative), and also produced realistic images and scenes by using 3D rendering platform, Autodesk Revit and Enscape3D platforms (see Software availability statement for alternatives). The final phase concerned dissemination, which is devoted to the distribution of information and knowledge to the general public through establishing new documentation methods in architectural conservation and built-heritage preservation, i.e., VR and Website Heritage Documentation (WHD).15\n\nAll of (A), (B), and(C) were gathered from open access sources, and available through institutes and their official websites.\n\nThe city of Alexandria was founded by Alexander the Great and early on featured a fusion of communities â Muslims, Christians, Jews, Armenians, Greeks, and Italians. It was once considered the jewel of the Mediterranean. Within the city, a case study was selected among sites with remarkable character. The selected case study is visible from the main roads in the area and represents a heritage style that has been understudied in the literature. Located at 52 Fouad Street, Alexandria, Egypt, the Société ImmobiliÚre building was chosen as a case study for this research (see Figure 1-A and Figure 1-B). Fouad Street is one of Alexandriaâs oldest streets and the most powerful symbol of Alexandriaâs remarkable history, with its elegant villas and antique shops. The Société ImmobiliÚre building is one of the Sigma Properties Companyâs most famous acquisitions. Indeed, the Sigma Properties Company saw the Société ImmobiliÚre building as an excellent investment opportunity. Designed in a unique Neo-Renaissance architectural style, the property has been listed as âitem 33â on the Heritage List and registered in the National Urban Coordination System Volume 2007 (see Figure 1-A).16,17\n\nThe Greek architects N. Paraskevas and P. Gripari designed the building. The Italian Averino family hired them after problems had emerged with their previous architect Giacomo Loria.18,19 The eclectic revivalist style of the late nineteenth century expressed perfectly the architectural pluralism and pro- European cosmopolitanism that prevailed in the city at the time. Later, between the two world wars, the decorative and early modern international styles became the most favoured architectural expressions in the city.19,20 The building under study was designed according to the popular eclectic aesthetics of the Neo- Renaissance style, and the building boasts a distinctive dome that can be seen from a distance (see Figure 1-C). The building has been used as a meeting chamber for Alexandriaâs elites and still stands out among many residences as a testimony to the wealth and pluralism of the cityâs cosmopolitan identity. The following table (Table 1) summarises general information about the Société ImmobiliÚre building.19,21\n\nThis study developed an application that seeks to digitally document one of Alexandriaâs most distinguished architectural heritage assets, exemplifying the rich architectural heritage found in Egypt. This application of HDPP (documentation phase, representation phase, rendering phase, and dissemination phase) creates a digital library to document heritage buildingsâ assets using the best suited available techniques to introduce an interactive 3D representation of the building and to model the building.\n\nImage-based techniques were used to gather data using photography and panoramic photography for the building as a whole and close-range photogrammetry for specific motifs. The tools used are as follows (see Figure 2-A):\n\n⢠DSLR camera and smartphone.\n\n⢠Professional camera tripod.\n\n⢠Padcaster tripod dolly wheels.\n\n⢠DJI Osmo Mobile 3 Gimbal for the smartphone.\n\nOther sources of information were used, where researchers contacted Alex Med of Bibliotheca Alexandrina, Sigma Properties Company, and the Cityâs Municipal of Alexandria, Egypt, to gather data for the Société ImmobiliÚre building under the regulations of registration in the National Urban Coordination System (see Figure 1-A), its location map in 1938 (see Figure 1-B), and historic photo for the building (see Figure 1-C) as reference documents for it. Moreover, the researchers surveyed and drew the Société ImmobiliÚre building indoors and outdoors as plan drawings of it (see Figure 2-B and Figure 2-C). All the above served to obtain information about the building from the time of its construction to the present day. The following table (Table 2) summarises the documentation phase for the Société ImmobiliÚre building.\n\nAfter the data have been gathered, they were digitised as a 3D point cloud. This 3D point cloud represents the data about the Société ImmobiliÚre building that was obtained during the documentation phase. Next, the representation phase using a photogrammetry platform follows these sequential steps:\n\n⢠Photos, videos, and panoramas were imported (3576 images) as 2D data into the RealityCapture platform (see Figure 3-A). Images were aligned in order to align 2D data and register the images. This process ended with the creation of 3D point cloud data in the form of one single component.\n\n⢠The alignment of image processing may be duplicated because it may not produce one single component upon the first trial. This process led researchers to create âcontrol pointsâ as 1D data on the 2D data to add more detailed information. This process was duplicated until the 3D point cloud data become a single component (see Figure 3-B-1).\n\n⢠The reconstruction process means that the component is reconstructed as a mesh model using graphics hardware. At this point, we had two options: a ânormal detailâ or a âhigh detailâ to reconstruct the component. In this study, the researchers choose the âhigh detailâ option, since it gives a more accurate meshing details, although it requires a longer time (see Figure 3-B-2).\n\n⢠The simplification process is the process by which the modelâs mesh size is optimised by reducing the triangle count. Once the researchers in this study were satisfied with the appearance of their reconstruction, it is good practice to define the overall triangle target for the 3D experiences. This practice represents the fidelity researchers seek to achieve (see Figure 3-B-3).\n\n⢠The texturing process uses source images to create a detailed 3D textured map and coloured triangle mesh (see Figure 3-C). The export mesh process can then export the project in many different file formats. We exported as an OBJ file for this workflow. OBJ file is a safe and standard format, although FBX file and GLB file are also good options.\n\nThe photos (A), (B), and(C) were acquired from the researchersâ working on the Société ImmobiliÚre building by using RealityCapture platform.\n\nThe following table (Table 3) summarises the representation phase for the Société ImmobiliÚre building.\n\nThe rendering phase using BIM and 3D rendering platforms follows these sequential steps:\n\n⢠3D textured and coloured triangle mesh is imported on the Autodesk Revit platform to document and model the Société ImmobiliÚre building (see Figure 4-A).\n\n⢠A Revit Family (RFA) is created. It is a unique library of BIM parametric objects (templates and 3D models of various objects i.e., doors, windows, stairs and so on) created by researchers on the Autodesk Revit platform, and it is used to document and model the Société ImmobiliÚre building (see Figure 4-B).\n\n⢠The Société ImmobiliÚre building is rendered on Autodesk Revit and Enscape3D platforms (see Figure 4-C).\n\nThe photos (A), (B), (C), and (D) were acquired from the researchersâ working on the Société ImmobiliÚre building by using Autodesk Revit and Enscape3D platforms.\n\nThe documenting and modelling of the Société ImmobiliÚre building were accomplished by using the Autodesk Revit platform and creating an RFA, a unique library of BIM parametric objects. During the rendering phase, we create a visualisation environment for the Société ImmobiliÚre building by visualising the resulting documentation model that illustrates the Société ImmobiliÚre buildingâs architectural style. Figure 4-D shows the rendered elevations using the Enscape3D platform which are the researchersâ work of this study; these elevations represent output to document the Société ImmobiliÚre building. This phase culminated with the creation of an HBIM model, offering a database and digital library about the Société ImmobiliÚre buildingâs assets.\n\nThe following table (Table 4) summarises the rendering phase for the Société ImmobiliÚre building.\n\nWe created new documentation methods for the Société ImmobiliÚre building. The VR and WHD both provide access to the documented HBIM model and its associated information and improve the process of documenting real estate intended for the real estate inventory lists of Egyptian Properties Protected from Demolition - Law No. 144 of 200622 and its executive regulations. These two methods allow users to fully know the documented database and digital library that include information about the assets of documented heritage buildings. Furthermore, the Société ImmobiliÚre building is preserved over time based on its HBIM model and digital technologies.\n\nThe dissemination phase constitutes the final phase in the HDPP and entails the dissemination of information about heritage to the public. As part of our case study, we created two methods to disseminate digital documentation about the Société ImmobiliÚre building.\n\nWe first created a VR for the Société ImmobiliÚre building. This VR experience uses the placement of the HBIM model to build a scene within the Enscape3D platform, integrating motion control and interactions in the Enscape3D platform. Finally, the immersive and interactive visualisation of the Société ImmobiliÚre building was integrated in the VR system. The setup for the VR system was as follows:\n\n⢠We installed a smartphone in a VR headset (Shinecon VR headset SC-G04, used in this research) and connected the headphones with a smartphone through Bluetooth (see Figure 5-A).\n\n⢠We mirrored the stream of the HBIM model in Real-Time (RT) rendering on the Enscape3D platform from a PC/laptop to a smartphone through the Letsview platform, a free wireless screen mirroring platform (see Figure 5-B).\n\n⢠We used a wireless controller (Microsoft Xbox One Wireless Controller, used in this research) to navigate the HBIM model in RT rendering on the Enscape3D platform (see Figure 5-C).\n\n⢠We modified the settings in the VR for the Société ImmobiliÚre building by entering the âsettingsâ tab and changing the âvisual,â âinput,â âdevices,â and âperformanceâ sub-tabs (see Figure 5-D).\n\nThe photos (A), (B), (C), (D), and (E) were acquired from the researchersâ working on the Société ImmobiliÚre building by using Enscape3D platform.\n\nOur second method was to create a website for the Société ImmobiliÚre building, by using the placement of the HBIM model to build a scene within the web version of the Enscape3D platform, to integrate motion control and interactions in the web version of the Enscape3D platform, and finally, to create an immersive and interactive visualisation of the Société ImmobiliÚre building in RT rendering. The settings for the Société ImmobiliÚre buildingâs website can be modified by clicking on the âsettingsâ tab and modifying the âvisualâ and âinputâ sub-tabs (see Figure 5-E).\n\nThe following table (Table 5) summarises the dissemination phase in the Société ImmobiliÚre building study.\n\n\nResults and discussion\n\nIn this research, we created new documentation methods and applied them to the Société ImmobiliÚre building. Based on the Société ImmobiliÚre building, VR and WHD were used to provide access to the documented HBIM model and its corollary information and improve the process of documenting real estate intended for the inventory lists of Egyptian Properties Protected from Demolition - Law No. 144 of 2006 and its executive regulations. These two methods through the application of HDPP lead to the creation of a digital database about the Société ImmobiliÚre building that allows users to acquire full knowledge about the documented database and digital library, which feature heritage building assets, thus also strengthening usersâ attachment to the place. Furthermore, the Société ImmobiliÚre building is revitalized over time based on the HBIM model and digital technologies (RealityCapture, Autodesk Revit, and Enscape3D platforms). The database result from the application tends to digitalise heritage buildings in 3D format and create digital libraries for those buildings. These new documentation methods open new possibilities for the documentation of heritage buildings and for new strategies for the preservation of Alexandriaâs architectural styles. Furthermore, academics and architects can deepen their knowledge about different design theories through the dissemination of these new documentation methods.\n\n\nConclusions\n\nThe research uses the implementation of HDPP and the usage of new documentation methods to communicate with architectural heritage data using image-based techniques accompanied by BIM, photogrammetry, and 3D rendering platforms to document heritage that tends to have a suitable documentation system for heritage buildingsâ assets. HDPP facilitates architectural research by creating a digital database based on video and image survey data, historical drawings and municipal maps, plan drawings, and reference books. The digital database helps better understand design theories through the on-site methods that highlight the history, architectural features, and styles of buildings, which increases the value of the heritage buildings.\n\nThe documentation methods, i.e., VR and WHD, enhance the availability of modelling and advanced curricular resources, allowing for global participation in the dissemination of digital documentation about heritage buildings.\n\nThis research uses the Société ImmobiliÚre building as a case study that was a starting point for applying new documentation methods in Alexandria, Egypt. Our methods document this building and create a digital path to help strengthen its image and connect the place to users, recreational areas are created to communicate and explore the cityâs architectural history. In addition, these documentation methods facilitate the preservation of heritage buildings using digital technologies and serve as a digital academic reference for the heritage buildings of Alexandria, Egypt.\n\n\nData availability\n\nMendeley: HDPP, Société ImmobiliÚre building, Alexandria, Egypt, https://data.mendeley.com/datasets/jy43tptxzz/2.23\n\nThis project contains the following underlying data:\n\n⢠RC (1).png (A figure shows aligning images processed through the RealityCapture platform to form 3D point cloud data as one component).\n\n⢠RC (2).png (A figure shows the reconstruction process through the RealityCapture platform to form one component as a mesh model).\n\n⢠RC (3).png (A figure shows texturing process through the RealityCapture platform to create a detailed 3D textured map and coloured triangle mesh â 01).\n\n⢠RC (4).png (A figure shows texturing process through the RealityCapture platform to create a detailed 3D textured map and coloured triangle mesh â 02).\n\n⢠REVIT (1).png (A figure shows a rendering of the Société ImmobiliÚre Building on the Autodesk Revit platform â 01).\n\n⢠REVIT (2).png (A figure shows a rendering of the Société ImmobiliÚre Building on the Autodesk Revit platform â 02).\n\n⢠REVIT_RC (1).png (A figure shows importing 3D textured and coloured triangle mesh on Autodesk Revit platform to documentation and modelling of Société ImmobiliÚre Building â 01).\n\n⢠REVIT_RC (2)-VocXt9.png (A figure shows importing 3D textured and coloured triangle mesh on Autodesk Revit platform to documentation and modelling of Société ImmobiliÚre Building â 02).\n\n⢠VR01.png (A figure shows modifying the VR settings on the Enscape3D platform for the Société ImmobiliÚre Building â Visual sub-tab).\n\n⢠VR02.png (A figure shows modifying the VR settings on the Enscape3D platform for the Société ImmobiliÚre Building â Input sub-tab).\n\n⢠VR03.png (A figure shows modifying the VR settings on the Enscape3D platform for the Société ImmobiliÚre Building â Devices sub-tab).\n\n⢠VR04.png (A figure shows modifying the VR settings on the Enscape3D platform for the Société ImmobiliÚre Building â Performance sub-tab).\n\n⢠WEB01.png (A figure shows modifying the settings for the Société ImmobiliÚre Building on the web version of the Enscape3D platform â Visual sub-tab).\n\n⢠WEB02.png (A figure shows modifying the settings for the Société ImmobiliÚre Building on the web version of the Enscape3D platform â Input sub-tab).\n\n⢠XBOX E.png (A figure shows navigating the HBIM model on the Enscape3D platform by using Microsoft Xbox One Wireless Controller).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\n\n\n⢠RealityCapture free alternatives: COLMAP and Meshroom platforms.\n\n⢠Autodesk Revit free alternative: TAD platform.\n\n⢠Autodesk Revit and Enscape3D free alternatives: Blender and LuxCoreRender platforms.",
"appendix": "Acknowledgments\n\nThe authors want to thank all the colleagues at the Department of Architectural, Faculty of Fine Arts, Alexandria University, Arab Academy for Science and Technology, Alex Med of Bibliotheca Alexandrina, Sigma Properties Company, and the Cityâs Municipal of Alexandria, Egypt, for being helpful and supportive throughout the process of this research.\n\n\nReferences\n\nUNESCO Digital Library: Charter on the Preservation of the Digital Heritage; 2009.2009; 33(October 2003): 0â4.Reference Source\n\nStone R, Ojika T: Virtual heritage: What next? IEEE Multimed. 2000; 7(2): 73â74. Publisher Full Text\n\nBeraldin JA, Blais F, Boulanger P, et al.: Real world modelling through high resolution digital 3D imaging of objects and structures. ISPRS J. Photogramm. Remote Sens. 2000; 55(4): 230â250. Publisher Full Text\n\nGillner S, Mallot HA: Virtual Reality and Spatial Cognition. Int. Encycl. Soc. Behav. Sci. 2001 Jan 1; 16211â4. Publisher Full Text Reference Source\n\nIED Barcelona Escola Superior de Disseny: MASTER IN DESIGN FOR VIRTUAL REALITY.Reference Source\n\nEze AB: Digitization of Archival Collections. Africa Sch Commun Issues. Strateg Challenges Libr. Philos. Pract. 2011.Reference Source\n\nLópez FJ, Lerones PM, Llamas J, et al.: A review of heritage building information modeling (H-BIM). Multimodal. Technol. Interact. 2018; 2(2). Publisher Full Text\n\nHichri N, Stefani C, De Luca L, et al.: Review of the « As-Built BIM » Approaches. Int Arch Photogramm Remote Sens Spat. Inf. Sci. 2013; XL-5/W1(February): 107â112. Publisher Full Text\n\nFai S, Graham K, Duckworth T, et al.: Building Information Modeling and Heritage Documentation. Autodesk Res. 2011; (June): 1â8.\n\nChevrier C, Charbonneau N, Grussenmeyer P, et al.: Parametric Documenting of Built Heritage: 3D Virtual Reconstruction of Architectural Details. Int. J. Archit. Comput. 2010; 8(2): 135â150. Publisher Full Text\n\nAnil EB, Tang P, Akinci B, et al.: Deviation analysis method for the assessment of the quality of the as-is Building Information Models generated from point cloud data. Autom. Constr. 2013; 35: 507â516. Publisher Full Text\n\nMurphy M, Mcgovern E, Pavia S: Historic building information modelling (HBIM). Struct. Surv. 2009; 27(4): 311â327. Publisher Full Text\n\nDore C, Murphy M: Semi-automatic generation of as-built BIM façade geometry from laser and image data. J. Inf. Technol. Constr. 2014; 19(February 2014): 20â46.\n\nRonnholm P: Registration quality - towards integration of laser scanning and photogrammetry. Off. Publ. - EuroSDR. 2011; 59: 6â43.\n\nDocumentation H: HERITAGE DOCUMENTATION - 3D MAPPING & 3D MODELING & SPATIAL ANALYSIS.2019.Reference Source\n\nAlexandrina B: Projects & Activities. Bibliotheca Alexandrina.2021. Reference Source\n\nAlexMed:Heritage Preservation Project. AlexMed - Bibliotheca Alexandrina. Alexandria; 2007. p. 155.\n\nEgypt G: Great Egypt: Egyptâs Belle Ãpoque architecture.Reference Source\n\nSigma Properties S.A.E. Societe Immobiliere: SIGMA PROPERTIES.2015.Reference Source\n\nAwad M: Conservation and Rehabilitation of Alexandriaâs City Center.Reference Source\n\nSabbagh M: Decision-making principles for re-developing heritage buildings - Towards a comprehensive framework in downtown Alexandria. AASTMT;2019.\n\nUrban Regeneration Project for Historic Cairo - UNESCO: Egyptian legislations in relation to the rehabilitation of Historic Cairo.2013; 8â10.Reference Source\n\nEl Menshawy A, Omar W, El Adawy S: HDPP, Société ImmobiliÚre building, Alexandria, Egypt - Mendeley Data. Mendeley Data.2022.Reference Source"
}
|
[
{
"id": "151718",
"date": "04 Oct 2022",
"name": "Osama Omar",
"expertise": [
"Reviewer Expertise BIM",
"Intelligent Buildings",
"Zero Energy",
"Smart Cities",
"Daylighting"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors,\nI would like to thank the author/s for their effort.\nA significant effort has been made by the authors to develop and implement Heritage Digitisation Process Phases (HDPP). Moreover, the authors establish new documentation methods in architectural conservation and built-heritage preservation, through using Virtual Reality (VR) and Website Heritage Documentation (WHD). But there are some minor comments that need from authors to modify it to enhance the research:\nIn the Introduction: it`s so brief and short. So, I prefer to expand the references in this area especially in Introduction with more updated references related to the topic specially when it come to the references its just 23 references which not enough to express solid base to built on it.\n\nIn Results and Discussion: Authors should explain the difficulties they encountered in documenting from an accurate point of view as well as the errors in the pictures taken and how the pictures are connected. Also, they need to give an idea about the timeline of all this process.\n\nConclusions: because it is so brief, there is need to express a coherent conclusion.\nBy the end, I want to say that you did a great job and all this comments is to encourage the author to enhance the quality of paper.\nThank you for your efforts.\nBest Wishes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9484",
"date": "22 Mar 2023",
"name": "Sherif El Adawy",
"role": "Author Response",
"response": "We would like to thank you, Dr. Osama Omar, for your comments. In replying to your comments, we took your comments into consideration for the new version of the article: All 23 references covered throughout the article are mostly up-to-date in the research's field, and the use of hyperlinks has been recommended by the editor to help readers find up-to-date information on terminologies, organizations, platforms, laws, books, people, and tools. Moreover, we added various tools related to photogrammetry and digital archiving in the introductory section, which can be divided into 5 classes, and we selected DSLR cameras and smartphones that will be used in the case study for the documentation phase of HDPP.  In the âResults and Discussionâ section, we clarified the difficulties we faced along the phases, which were concentrated at the first two phases, and also mentioned the phases' timeline for implementing the HDPP on the Société ImmobiliÚre building.  In the âConclusionsâ section, we listed the advantages and disadvantages of what we learned from our experiences in this research and recommended solutions for the disadvantages that could be taken into consideration and used in the future."
}
]
},
{
"id": "152650",
"date": "13 Oct 2022",
"name": "Nabil Ibrahim Mohareb",
"expertise": [
"Reviewer Expertise Spatial configuration analysis for historical cities",
"photogrammetry",
"movement analysis."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper offers an interesting digital archiving method for one of Alexandria's well-known buildings. The paper is well organized. My comments are a mix of technical and general thoughts.\nIt is preferable to define the designated era in the abstract section, as it is not evident in the text â... there is no suitable documentation system for these more recent assets.â\n\nIn the introduction, it was difficult to spot the research aim directly. I think this is the main focus/aim of the research â... Therefore, it is essential to introduce technical and historical approaches in both the BIM environment and the point clouds, to model the different virtual parametric components and achieve a BIM model for the architectural heritage under analysis.â I would strongly advise you to emphasize this in both the abstract and the introductory sections.\n\nIn the literature review section, other photogrammetry and digital archiving strategies (methods/approaches) could be compared (briefly) with the recommended methods and tools to demonstrate that your suggested tools are more efficient.\n\nâ... Obtaining the parametric building elements from the 3D point cloud is a time-consuming and error-prone manual process because no automation or platform processes currently exist that can ensure a direct change from the 3D point cloud to full BIM models.â If this is the main research gap, it is preferable to indicate so explicitly in the introduction.\n\nYour readers may benefit from a definition of technical phrases such as 3D point cloud.\n\nThere is no mention of ground sampling distance (GSD). It is essential to understand how your model gets scaled. When photogrammetric techniques are utilized, metric data cannot be disregarded. Please elaborate on technical concerns pertaining to the camera and image information.\n\nIt would be informative to specify whether the final 3D model reflects only the building's two façades or the entire building.\n\nIn the conclusion section, I would propose highlighting any potential limits of this technique and comparing it to other methods (refer to my earlier comment) to highlight its advantages and disadvantages.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9485",
"date": "22 Mar 2023",
"name": "Sherif El Adawy",
"role": "Author Response",
"response": "We would like to thank you, Dr. Nabil Ibrahim Mohareb, for your comments. In replying to your comments, we took your comments into consideration for the new version of the article: We defined the designated era in the abstract section: âIn Alexandria, the heritage value of the city has decreased since the beginning of the third millennium of the Common Era because there is no suitable digital documentation system for these more recent assets.â  We explained the aim of the research in the abstract: âIn this research, we primarily seek to implement Heritage Digitisation Process Phases (HDPP) by introducing both the Building Information Modelling (BIM) environment and the point clouds for achieving a Historic Building Information Modelling (HBIM) model and to establish new documentation methods in architectural conservation and built-heritage preservation, i.e., Virtual Reality (VR) and Website Heritage Documentation (WHD).â And in âResearch aimâ section, we clarified the creation of the digital database was based on technical and historical approaches in both the BIM environment and the point clouds, using video and image survey data, historical drawings and maps of the municipality, plan drawings, and reference books.  We mentioned the tools related to photogrammetry and digital archiving and selected the tools we used in the case study.  Throughout these words, \"Obtaining the parametric building elements from the 3D point cloud is a time-consuming and error-prone manual process because no automation or platform processes currently exist that can ensure a direct change from the 3D point cloud to full BIM models\", we wanted to figure out that there is no direct platform to convert the 3D point cloud to a BIM model, as well as that we took the 3D point cloud as a reference to build up the BIM model, which took us a period of time to execute. Referring to the main research gap, this was not the main research gap for this article, but we would consider this research gap in our consideration for our next article.  All technical terms, such as the 3D point cloud, have been hyperlinked, which was recommended by the editor to help readers find up-to-date information on terminologies, organizations, platforms, laws, books, people, and tools.  In terms of ground sampling distance (GSD), we did not use a drone as a photogrammetric technique in our article, instead employing a new method that included a DSLR camera, a smartphone, a professional camera tripod, a padcaster tripod dolly wheels, and a DJI Osmo Mobile 3 Gimbal for the smartphone.  We indicated that the final 3D model is for the entire buildingâs façades by adding the word \"entire.\"  We have covered the advantages and disadvantages of what we got out of our experiences in the conclusion section."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1044
|
https://f1000research.com/articles/11-1008/v1
|
07 Sep 22
|
{
"type": "Research Article",
"title": "The coronavirus disease 2019 (COVID-19) pandemic in nursing homes â the experience of care workers in Poland",
"authors": [
"Ryszard Necel"
],
"abstract": "Background: Nursing homes in Poland are the most common formal care institutions for dependent people. During the coronavirus disease 2019 (COVID-19) pandemic, nursing homes had particularly high infection rates. In this context, it is important to ask about the experiences of the care workers working in these institutions. Methods: This research was conducted using the computer-assisted web interviewing (CAWI) technique in five provinces in Poland. The field research was carried out in April 2021. The research sample included, among others, nurses, care workers, therapists, social workers and the management staff of institutions whose representatives worked during the COVID-19 pandemic. Respondents were asked to assess the care provided to residents. Results: It turned out that the vast majority of respondents positively assessed the fulfillment of the basic living needs of residents and the availability of care. The assessment of the organization of residentsâ leisure time, the fulfillment of their religious and cultural needs, and the issue of maintaining contacts with the social environment was less satisfactory. The article also describes the results of care institution employeesâ self-assessment of their mental health. For the majority, the most stressful factor was the need to work in a health-threatening environment and the sense of responsibility for the residents. Regarding the availability of the forms of support offered to workers experiencing deteriorating mental health due to working in the pandemic situation, more than a third said that their institution did not offer any form of assistance. Conclusions: The article lists a number of recommendations. In the light of the data obtained, it is necessary to increase the intensity of services provided to residents of nursing homes in terms of organizing their free time, meeting their religious and cultural needs and maintaining contact with the social environment.",
"keywords": [
"nursing homes",
"social care",
"care workers",
"culture of disaster",
"mental health"
],
"content": "Introduction\n\nNursing homes constitute the largest number ofall care institutions in Poland. These facilities are for people who require round-the-clock care due to age, illness or disability and cannot be provided with the necessary help in the form of community care services. Nursing home residents also suffered the most from the health consequences of the coronavirus disease 2019 (COVID-19) pandemic. By the end of 2020, over a third of nursing home residents (33.9%) had contracted coronavirus (Statistics Poland, 2021). For comparison, in other 24/7 inpatient facilities, the incidence rate was much lower. For example, 10.9% of all residents of family nursing homes and 4.1% of all residents of homeless shelters contracted the virus (Statistics Poland, 2021). However, it should be emphasized that the Central Statistical Office, a government administration agency dealing with collecting and sharing statistical data on most areas of public life, does not publish official data on morbidity among medical personnel and care workers. The lack of this type of information significantly limits the ability to objectively assess the effects of the pandemic on the capacity and effectiveness of the care system. Nevertheless, both in Poland and throughout Europe, the evidence-based approach has been a dynamically developing perspective in research on the health of the population and its individual subgroups since the 1990s (Raine, 1998; Niessen et al., 2000).\n\nThe conducted empirical research was to partially fill the gap in knowledge about the condition of care workers in Poland during the coronavirus pandemic. However, we have consciously gone beyond the narrow analysis of public statistics and taken account of the social aspect of the pandemic. This research is based on the culture-of-disaster perspective (see, among others, Revet, 2020; Revet & Langumier, 2015; Bankoff, 2003; Medina, 2016; Benadusi, 2014), which focuses on âwhat disaster is made of for each of the actors involvedâ (Revet & Langumier, 2015). The subject of interest is the attitudes toward the crisis situation shared by individuals, including how they experienced the reality of the pandemic. Etienne Wenger adds that the relations between the functioning of asocial system and the orientations toward it are shaped within the community of practice (Wenger, 1998). These practices are revealed not only in action, but also through the shared knowledge, attitudes and beliefs that create a certain common view of the world (Wenger, 1998, p. 47).\n\nIt can therefore be asked why, in the proposed cultural approach, the experiences of care workers should be considered important? Firstly, the knowledge of practitioners should be treated as a social mirror of the resourcefulness of institutions in the crisis caused by the pandemic. The capacity to deliver care services in a pandemic situation may be extended along the continuum between two poles as defined by Nilsson and Olaison: suspension versus seeking new solutions (Nilsson & Olaison, 2020). Of course, care institutions fulfilled their basic functions despite significant difficulties in operating during the successive waves of the pandemic. However, it is worth referring to the experience of practitioners and asking about the ability to meet the caring, activating, religious and other needs of nursing home residents during the pandemic. This question is answered in the first part of the article, which presents nursing homes employeesâ assessments of the individual assistance measures.\n\nSecondly, the analysis of employeesâ experiences may become the basis for formulating recommendations on changes in the care system. The previous analyses are dominated by the belief that the pandemic should be treated as a wake-up call (Fischer et al., 2020) in the face of the challenges of an aging society. The academic discourse calls for necessary reforms to ensure high-quality care for people with limited independence in the future (Daly, 2020). When planning social policies (Rothman, 2007), it is worth listening to the needs of those who faced the consequences of the pandemic on a daily basis. Based on the analysis of expectations and preferences regarding the protection of mental health, a proposal for changes is presented that would enable better preparation for possible crisis situations caused by the need to work in an emergency.\n\nTo sum up, the research carried out in the group of care workers allows the voice of those whose positions in the public discourse are not properly articulated to be captured. On the one hand, they do not have enough time to shape their own narrative in the public discourse as they simply deal with duties related to care and its organization. During the pandemic, this care is even more demanding than before (Fallon et al., 2020; Rodrigues et al., 2021). On the other hand, care workers frequently lack representation because they have a âsilent voiceâ due to poorly organized trade unions and employee organizations in comparison with the healthcare sector, among others (Daly, 2020).\n\nThe main objective of this article is to analyze the attitudes of care workers in nursing homes toward the reality of the pandemic in Poland, in four basic contexts: assessment of the degree of social care in the pandemic era; assessment of the most stressful factors since the outbreak of the pandemic; assessment of the mental wellbeing of care workers; and evaluation of institutional measures taken to improve mental health. More specifically, the following hypotheses are tested: 1) the assessment of the degree of social care in the pandemic era differs depending on the type of municipality represented by the respondent (urban, rural, urban-rural), the experience of care workers (measured by seniority), the type of position held (managers or frontline workers) and the type of nursing home (nursing homes for people with mental disorders, nursing homes for people with reduced mobility, nursing homes for the elderly); 2) the assessment of the most stressful factors since the outbreak of the pandemic differs depending on the type of municipality represented by the respondent, the experience of care workers, the type of position held and the type of nursing home; 3) the assessment of the mental wellbeing of care workers differs depending on the type of municipality represented by the respondent, the experience of care workers, the type of position held and the type of nursing home; and 4) the evaluation of institutional measures taken to improve mental health differs depending on the type of municipality represented by the respondent, the experience of care workers, the type of position held and the type of nursing home.\n\n\nMethods\n\nThe field research was carried out in April 2021. It was conducted using the computer-assisted web interviewing (CAWI) technique. The CAWI questionnaire consisted of 19 questions grouped in following topics: mental well-being of employees, internal rules of the functioning of the institution, residentsâ needs, cooperation with the environment, birth certificate questions. The questionnaire can be found as Extended data (Necel, 2022). The questionnaire hasnât been validated.\n\nThe CAWI technique was chosen because it provides easy access to the population, which was particularly important during the pandemic due to the need to maintain social distancing. Moreover, this tool allows the avoidance of open-ended questions and questions formulated primarily in the form of statements, making it useful in this project. The link to the questionnaire, along with a covering letter, was sent to the secretariats of nursing homes with a request that two care workers fill in the questionnaire, so that there is no imbalance in the number of questionnaires received between institutions with different numbers of employees. The inclusion criteria (verified by the answers given to the corresponding survey questions) included being engaged in work during the COVID-19 pandemic to April 2021. The exclusion criterion was being an employee who was not a care worker, e.g. an accountant.\n\nThe Ethics Committee for Research Involving Human Participants at Adam Mickiewicz University in Poznan granted an approval of the research project (Resolution No. 4/2021/2022 adopted on 25 January 2022). Each research participant signed a voluntary and informed consent form for participation in the research. We uploaded âconsent formâ to a repository.\n\nThe research used a non-probability purposive sampling asit was carried out on a relatively small population with a well-known structure with the possibility of accessing all its representatives.\n\nThe research was carried out in all nursing homes in five provinces in Poland (there are a total of 16 provinces in Poland): 54 nursing homes in Lower Silesia, 55 in the Lodz Province, 47 in the Subcarpathian Province, 44 in the Lublin Province and 62 in the Greater Poland Province. The provinces were selected for the research based on the analysis of the following variables: the number of nursing homes, the number of places in these homes, the number of residents and the number of employees. On this basis, provinces with similar care resources were selected. The employee population involved: management staff, administration workers and care workers, which included social workers, nurses and therapists, that is, representatives of the profession providing direct support and help to residents of nursing homes.\n\nA chi-square test (with Yatesâ correction for 2Ã2 tables) was used to compare qualitative variables among the groups. In the case of low values in the contingency tables, Fisherâs exact test was used instead. The level of significance for all statistical tests was set at 0.05. Thus, all p values below 0.05 were interpreted as showing significant correlations. R 4.0.5 was used for the computations (RRID:SCR_001905).\n\n\nResults\n\nBy the end of 2020, 37,309 care workers were employed in Poland. In the five voivodships surveyed, a total of 18,659 carers were employed. The research was conducted among 189 of them (Ministry of Family and Social Policy, 2022). Employees representing three types of care facilities participated in the research: nursing homes for people with mental disorders â 36.5%, nursing homes for people with reduced mobility â 36.5%, and nursing homes for the elderly â 27.0%. The nursing homes whose employees took part in the research were located primarily in urban municipalities â 56%, followed by urban-rural municipalities â 19.8%, and rural municipalities â 24.2% (Necel, 2022).\n\nRespondents were most often care workers â 44.9% (of whom 18.6% were nurses) and slightly less often management staff â 29.9%, while 21.6% of all respondents were administration workers. As a rule, respondents had extensive work experience in the social welfare sector. Over one-third (36.2%) had more than 20 years of work experience. There was also a large group of employees with 10 to 19 years of work experience, who constituted 33.3% of all respondents. Only 11.9% of respondents had less than two years of work experience, and 18.6% of the surveyed nursing home employees had been employed for two to nine years.\n\nRespondents were asked to assess to what extent the basic needs of residents were met during the pandemic (Table 1). The question was accompanied by multiple-choice answers detailing the various activities of care institutions in this area. Most respondents assessed the fulfillment of the basic living needs of residents (food, clothing, hygiene products) as definitely good (78.3%) and somewhat good (20.6%). Similarly, the represented institutions were rated highly in terms of the availability of care and support services. The implementation of this function was assessed as definitely good by 64.6% of respondents, or as somewhat good by 32.8%. In other aspects of the functioning of the nursing homes, respondents had more diverse opinions. When it comes to organizing the leisure time of residents, 24.3% assessed it as definitely good, and 46.6% as somewhat good. On the other hand, 14.3% of respondents were of the opposite opinion and answered âsomewhat badâ or âvery badâ. Just over half of respondents (54.5%) assessed the fulfillment of religious and cultural needs as definitely good or somewhat good, and about one-fifth of respondents had the opposite opinion (22.8% â the cumulative percentage of answers âsomewhat badâ and âvery badâ). In the context of the restrictions introduced on visits by outsiders during the pandemic, it is surprising that 21.2% of respondents assessed activities related to maintaining and developing contact with family and the social environment as definitely good and as many as 42.9% as somewhat good. It is worth emphasizing, however, that over one-fifth of respondents had a different opinion (21.2% â the cumulative percentage of answers âsomewhat badâ and âvery badâ), and 14.8% of employees did not have an opinion on this subject. The activities of care institutions for the benefit of the local community were rated the least positively, as 5.3% answered âdefinitely goodâ, 18% âsomewhat goodâ, and as many as 55.6% of respondents did not express an opinion on this subject.\n\nThe statistical analyses conducted (chi-square test or Fisherâs exact test) showed that the assessments of the functioning of nursing homes during the pandemic were different depending on the respondentsâ job position. Significant differences were noted in the opinions regarding the fulfillment of religious and cultural needs (p < 0.001, probability value < 0.05), the implementation of which was assessed as definitely good (28.4%) and somewhat good (33.3%) by care workers, while only 2% of directors expressed an overwhelmingly positive opinion, and 38%a somewhat positive opinion about this aspect. The job position also differentiated the views on maintaining contact with the family and the social environment (p = 0.034, probability value < 0.05). In this case, care workers were also more optimistic in their assessments. This aspect was assessed as definitely good by 29.6%, or as somewhat good by 39.5%. Only 10% of management representatives gave a similar opinion, responding âdefinitely goodâ, and 40% responded âsomewhat goodâ. The job position was also important when assessing activities undertaken for the benefit of the local environment (p = 0.008, probability value < 0.05). As was the case with the above dependencies, this time care workers were also more satisfied than the management of the facility. While 8.6% of care workers assessed this function as definitely good and 17.2% as somewhat good, only 2% of the management staff assessed it as definitely good and14% as somewhat good.\n\nIn the course of the analyses, the hypothesis has been confirmed that the type of nursing home in which an employee works significantly differentiates the assessment of the functioning of the care institution in the context of meeting the basic needs of residents. A statistically significant relationship was noted in the area of providing care and support services (p = 0.009, probability value < 0.05). Namely, 73.9% of employees of nursing homes for people with mental disorders assessed this aspect of activity as definitely good. In the case of nursing homes for the elderly, 66.6% of answers were definitely good, while in the case of nursing homes for people with reduced mobility, this number was 53.6%. The differentiation also concerned the organization of leisure time for residents (p = 0.002, probability value < 0.05). Namely, 37.6% of respondents from nursing homes for people with mental disorders expressed a definitely good opinion about this form of support. Only 23.5% of the representatives of nursing homes for the elderly and 11.5% of respondents from nursing homes for people with reduced mobility were of a similar opinion. Significant differences were also noted in terms of maintaining and developing contact with family and the environment (p = 0.046, probability value < 0.05). They were assessed the best by the representatives of nursing homes for people with mental disorders (27.5% definitely good, and the worst by respondents from homes for people with reduced mobility (13% definitely good). The analyses of statistical dependencies did not show that the type of municipality or the respondentâs seniority statistically significantly differentiated the assessment of the functioning of a care institution.\n\nRespondents were asked about the most stressful factors at work since the declaration of the pandemic (Table 2). The surveyed nursing home employees most often indicated the necessity to work under the conditions of health risk as a stressful factor. This answer was given by 68.3% of respondents.\n\nAnother stressful factor for respondents was their responsibility for the residents of nursing homes, which was emphasized by 62.4%. Other stressors were mentioned less frequently. More than one-third of respondents (35.4%) mentioned combining work with duties toward their relatives, such as taking care of children and other dependent family members. In the light of the empirical data obtained, unclear procedures in the pandemic situation also turned out to be stressful at three levels: national, local and at the level of the institution where respondents worked. Nearly one-third of respondents pointed to the lack of clear procedures at the national level (32.8%). Interestingly, the lack of clear procedures at the local level was indicated as a stressful factor by 9.5% of respondents, while unclear procedures inside an institution were pointed out by 4.2%.\n\nResponsibility for others outside the workplace was a stressful factor for almost every fourth respondent (24.3%). However, for every fifth respondent (19%), stress and tension resulted from the limited possibility of testing for the presence of coronavirus. The lack of or too little personal protective equipment (PPE) in the workplace was selected as a stressor by only 4.2% of respondents.\n\nConflicts emerging due to tension in the workplace were stressful for 18% of respondents, while in their private life it was 2.1% and thus the least frequently chosen stress factor.\n\nThe analyses conducted (chi-square test and Fisherâs exact test) have confirmed the hypothesis that opinions about stressors differ depending on seniority. Statistically significant differences were noted in the opinions on combining work with responsibilities toward loved ones (p = 0.005, probability value < 0.05). This issue was mentioned by 57.5% of respondents with work experience from three to nine years, and only by 19% of those with work experience of up to two years. Seniority significantly differentiated the employees in their statements about unclear procedures at the local level (p = 0.026, probability value < 0.05), which was a source of stress mainly for respondents with up to two years of experience, among whom 28.5% pointed to this problem. However, this factor was stressful for only 9.3% of people with the longest work experience (20 years and more). The analyses have also confirmed the hypothesis about the relationship between the position held and the importance given to unclear procedures at the local level (p = 0.029, probability value < 0.05). This problem was much more often emphasized as stressful by administration employees (11.1%). None of the management staff representatives indicated the importance of this factor. However, the sense of responsibility for the residents (p < 0.001, probability value < 0.05) was much more likely to be a stressor for institution directors (88%) than for administration employees (47.2%) or care workers (56.7%).\n\nThere were no statistically significant correlations confirming the hypothesis about differences in the perception of stressors depending on the type of the municipality represented or the type of nursing home.\n\nThe question about stress factors at work corresponds to another research problem concerning the mental health of nursing home workers during the pandemic (Table 3). Respondents were asked a series of questions based on the following template âDid you ⊠in the months since the outbreak of the pandemic?â with selected areas of wellbeing clarified. They were asked to respond based on the following multiple-choice answers: âmuch more than usualâ, âslightly more than usualâ, âthe same as usualâ, âslightly less than usualâ and âmuch less than usualâ. Areas of particular interest were those that changed during the pandemic and so the cumulative percentages of the responses âmuch more than usualâ and âslightly more than usualâ are presented to describe the results.\n\nThe research results indicate the widespread experience of increased stress among nursing home employees. Over 84% of respondents felt more stressed than usual on a daily basis. The vast majority of respondents (over 70%) felt motivated to act at that time. Most respondents also experienced chronic fatigue (82%), and more than half had trouble sleeping (54%).\n\nThe intensity of neurotic reactions was also greater than usual. Almost 70% of research participants felt more anxiety or severe anxiety than usual. Over 60% of respondents felt increased nervousness and tension in contacts with people.\n\nThe studied individuals relatively less frequently manifested selected depressive reactions in the form of cognitive changes consisting of perceiving the situation as hopeless (less than 50%) and the inability to experience pleasure (slightly over 33%).\n\nThe hypothesis that psychological wellbeing is related to the position held has been confirmed by the analysis of statistical correlations in only one aspect, that is, the mobilization to act (p < 0.001, probability value < 0.05). It was felt most strongly by directors, as many as 68% of whom felt it much more than usual, and the weakest by administrative employees, only 25% of whom were mobilized to act much more than usual. It turns out that the differences in the mobilization to act were also influenced by the type of nursing home in which a respondent works (p = 0.013, probability value < 0.05). The mobilization function of the pandemic was higher in facilities for the elderly, where 45.1% of respondents felt this much more than before the pandemic, while in facilities for people with reduced mobility and mental disorders, it was 36.2% and 40.5%, respectively. The hypothesis that the type of municipality represented differentiates the psychological wellbeing of respondents has also been confirmed. Statistically significant differences were noted in the case of nervousness in contacts with others (p = 0.015, probability value < 0.05), which was felt much more than usual by nursing home employees from urban-rural municipalities (27.7%) than by those from rural (13.6%) or urban (7.8%) ones.\n\nIn the research, respondents were asked about solutions used in their workplaces to protect mental health (Table 4). For this purpose, respondents were asked the following closed-ended question: âWhat solutions have been implemented to protect the mental health of people working in your institution?â The following forms of support were distinguished among the responses: âconversation with a psychologistâ, âsupport groupâ, âsupervisionâ and âotherâ. Over one-third of respondents (37%) did not record any activities in their institution aimed at protecting mental health during the pandemic. This means they did not choose any of the possible answers. Among 63% of respondents who thought that their institution had implemented support instruments, most mentioned a conversation with a psychologist. Such a solution was observed by over one-third (35.4%) within their institution. According to some respondents, the institutions also organized support groups. This type of assistance was indicated by approximately one-fifth of respondents (21.7%). Moreover, 7.4% of respondents could have performed their work under supervision, which can also be classified as a solution aimed at protecting mental health. Finally, 11.1% of respondents indicated other types of support.\n\nThe analysis of statistical correlations performed using the chi-square test did not show significant differences in the opinions on the forms of psychological support provided depending on the position held, seniority, the type of nursing home and the type of municipality in which the facility was situated.\n\nRespondents were also asked the following question about their expectations toward the preferred forms of support for mental wellbeing: âWhat kind of activities aimed at protecting the mental health of employees would you expect in your institution?â Respondents had the opportunity to express their own expectations toward the represented institution. The empirical material collected was categorized into several responses.\n\nMost respondents (23.8%) expected a conversation with a psychologist. Several times respondents indicated that a psychologist should be from outside the institution, which may result from the reluctance to reveal oneâs thoughts to a person one knows due to the lack of anonymity. Support groups were another most frequently mentioned activity aimed at protecting the mental health of employees that, according to the surveyed persons, should have been organized in their institution (11.1%). Another category of answers related to rest or holidaysâ this was indicated by 6.3% of respondents. Every twentieth respondent (5.2%) indicated the need for the institution to organize training sessions and workshops to help them cope better in the pandemic situation. The same percentage of respondents indicated supervision (5.2%) as well as the need to employ additional people (4.8%). Other responses included expectations related to remuneration (higher salary, bonuses), the role of management (âclearer decisions of the managersâ, âsupport from the managersâ) as well as the bodies running the institutions (âinterest of the unit in chargeâ). There were also references to the situation outside the institutions. Respondents indicated that the following would be helpful: âclear procedures at the national levelâ or a change in the climate of public opinion, which one of the respondents expressed as: âthe end of the state of the media pandemic panicâ.\n\n\nDiscussion\n\nThe provision of care requires direct interpersonal contacts and spatial closeness (Weicht, 2015). Maintaining these conditions is a particularly difficult task in the era of the COVID-19 pandemic, where care workers help others and risk their own health (Devlieghere & Roose, 2020) and often their life, as indicated in some countries by higher mortality rates among care workers than in the entire population (Daly, 2020). In medical care, from the beginning, there was general agreement in the medical community to calculate the benefits of providing procedures that do not directly save life and health relative to the âcosts of contactâ, that is, the possibility of contracting the virus in contacts with the healthcare system (Bhatia et al., 2021). The costs of direct contact were also calculated in social care, as evidenced by the empirical data obtained. On the one hand, ensuring social activation of residents, concern for their spiritual development or integration with the local environment was associated with a higher risk of infection understood as a cost in this context, which could directly translate into a lower assessment of these forms of support. On the other hand, regardless of this cost, the implementation of basic rights, such as subsistence rights and security rights (Shue, 1980), can be considered satisfactory. This is evidenced by the high assessment of the fulfillment of living and care needs obtained regardless of the prevailing pandemic conditions. However, the employees of care institutions were relatively less satisfied with the implementation of freedom rights relating to the need for individual autonomy (Fabre, 2004). Consequently, activities in the areas of social activation, spiritual development and integration with the local environment, which required contacts with people and entities from outside the institutions, were assessed as less satisfactory. These could have been associated with a greater risk of infection, and the cost of care could have been treated as too high relative to profit.\n\nIn the context of questions about providing assistance to residents, despite the above-mentioned differences in assessments, it is worth emphasizing that positive assessments outweighed negative ones in each dimension of assistance activities. A particularly high assessment of the fulfillment of the living and care needs was possible thanks to the great commitment of care workers. Not without significance was the amendment to the Act on the posting of employees as part of the provision of services introduced in 2020, giving nursing home managers the ability to ask their employees to provide the necessary overtime work after obtaining their prior consent (Amendment of the act on the posting of workers as part of the provision of services and some other acts, 2020). This administrative decision made it possible to increase the number of care workers, who were seriously threatened by COVID-19 around the world (Comas-Herrera et al., 2020), including in Poland. It is worth highlighting that there were relatively fewer positive assessments when it comes to providing religious services to residents (Swift, 2020; Drummond & Carey, 2020). Pastoral care is of great importance for believers, especially when they experience suffering and illness (Swift, 2020), and spiritual support can improve wellbeing and physical condition (Koening, 2020). Therefore, cooperation with churches and religious associations is significant.\n\nAt the peaks of the successive waves of the coronavirus in Poland, which were in March and April 2020, October and November 2020, and in March and April 2021, nursing homes were closed to visitors or contact with outsiders was significantly limited. In this context, it is surprising that the activities aimed at maintaining and developing contact between residents and their families and relatives were highly rated. This is because during the lockdown, the institutionsâ employees tried to keep in touch with residentsâ relatives using new teleinformation technologies (mainly instant messaging). Despite the many risks and weaknesses associated with the use of new technologies: insufficient digital competences of users (de Jonge et al., 2020), a lack of trust in technology and discomfort in its use âprimarily among seniors (Swinford et al., 2020), p. 519), it is likely that new communication technologies will stay in social work and care for longer (Scheyett, 2020). Therefore, it is worth considering how to permanently include them in the mainstream of care and how to use them to build effective relationships with residentsâ relatives. The assessments of available forms of support presented in this article only took into account the perspective of care workers. Cognitively, it would be interesting to continue the research among the residents themselves using qualitative methods, such as individual in-depth interviews, to capture the experiences of people who require care and have to face the constraints imposed due to the pandemic.\n\nWorking in a situation where there is a high risk of losing health translates into weakened mental health (Khatri et al., 2019). This observation was also confirmed in the conducted research. Nursing home workers noted significant personal and emotional labor costs during the pandemic. The sense of responsibility for others, that is, residents of the nursing home and their own family members, was the dominant stress factor for them. The literature raises the issue of a specific conflict that may occur between the need to protect oneself and oneâs loved ones, and the sense of responsibility in the context of fulfilling the professional role of a care worker (Baum, 2012; Dekel & Baum, 2010). Additionally, working in a nursing home during the pandemic is an exemplification of the idea of âshared traumatic realityâ which consists of being subject to the same consequences of a crisis situation as the client (Cain, 2015). Consequently, this can lead to blurring boundaries between a worker and a client, as well as to greater emotional distress (Caroll et al., 2010).\n\nUnder conditions of stress and weakened mental health, activities supporting employees in coping with this type of problem are of great importance. On the one hand, administrative measures should be taken at the management level of a specific institution to increase work safety, such as increasing the availability of PPE, changing the organization of work, including delegating employees to other activities, or increasing the number of working hours. In this area, it is also important to create appropriate financial incentives (Garbers & Konradt, 2014; Landry et al., 2017) or compensation for work in extraordinary conditions. On the other hand, it is also necessary to take care of the emotional wellbeing of employees by providing them with appropriate support in the form of professional self-care (Skinner, 2015). In social work, the importance of professional self-care in counteracting distress and supporting a healthier, sustainable workforce is emphasized (Bloomquist, 2015). Hence the need to educate students of welfare professions in professional self-care (Newell & Nelson-Gardell, 2014) and to organize professional support of supervisors and psychologists for experienced care workers.\n\nThis research has several limitations. Firstly, the research sample included nursing home employees from five provinces in Poland. Therefore, care should be taken not to generalize (extrapolate) the research results to all care workers throughout the country. Secondly, the research was carried out using the CAWI technique, which was the only possible way to safely conduct the research during the pandemic. However, it limits the possibility of verifying whether a respondent was a nursing home employee actually working during the pandemic. Thirdly, the opinions expressed were undoubtedly influenced to some extent by the context in which the surveys were carried out. This was when the respondents were experiencing the third wave of the pandemic. On the one hand, they were not sure how long the crisis situation would last and what its far-reaching consequences would be for the functioning of the institution and its clients. On the other hand, they already had a certain sense of security guaranteed by vaccinations provided to all care workers and residents.\n\n\nConclusion\n\nIn this research, stressors have been identified and changes in the mental wellbeing of nursing home workers during the pandemic have been described. An in-depth analysis of this data makes it possible to design and implement forms of support most suitable for the reported needs, based on the direct experiences of care workers. The organization of various forms of mental health support for formal care workers, including nursing staff, is the main challenge facing the social welfare system in Poland. The offer should include individual psychological help (provided by a psychologist from outside the institution), supervision and peer support groups. It is also necessary to monitor the effectiveness of activities aimed at protecting the mental health of employees (continuous evaluation) and introduce possible modifications in order to increase their effectiveness. In the light of the data obtained, it is also necessary to increase the intensity of services provided to residents of nursing homes in terms of organizing their free time, meeting their religious and cultural needs and maintaining contact with the social environment. In this context, it is necessary to develop innovative (in Poland) forms of support, such as laughter therapy (Kuru & Kublay, 2017) or meditation-relaxation strategies (Lindberg, 2005), which not only support spiritual growth and reduce negative emotional states, but are also an interesting way of spending free time. It is worth noting that this type of activity can be conducted remotely using video communication apps.\n\n\nData availability\n\nOpen Science Framework: The Coronavirus Pandemic in Nursing Homes â the Experience of Care Workers in Poland, https://doi.org/10.17605/OSF.IO/72NM4 (Necel, 2022).\n\nOpen Science Framework: The Coronavirus Pandemic in Nursing Homes â the Experience of Care Workers in Poland, https://doi.org/10.17605/OSF.IO/72NM4 (Necel, 2022).\n\nThis project contains the following extended data:\n\n⢠Questionnaire\n\n⢠Cover letter\n\n⢠Consent form\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgments\n\nI am thankful to Regional Center for Social Policy in PoznaÅ for suport to conducting a research and investigation process.\n\n\nReferences\n\nAmendment of the act on the posting of workers as part of the provision of services and some other acts:Dz.U. 2020.1423 (POL).2020.Reference Source\n\nBankoff G: Cultures of disaster. Society and natural hazard in the Philippines. Routledge Curzon;2003.\n\nBaum N: âEmergency Routineâ: The Experience of Professionals in a Shared Traumatic Reality of War. Br. J. Soc. Work. 2012; 42(3): 424â442. Publisher Full Text\n\nBenadusi M: Unpacking Culture in DRR Education. J Contingencies Crisis Man. 2014; 22: 174â183. Publisher Full Text\n\nBhatia RS, Shojania KG, Levinson W: Cost of contact: redesigning healthcare in the age of COVID. BMJ Qual. Saf. 2021; 30: 236â239. PubMed Abstract | Publisher Full Text\n\nBloomquist KR, Wood L, Friedmeyer-Trainor K, et al.: Self-care and Professional Quality of Life: Predictive Factors among MSW Practitioners. Adv. Soc. Work. 2015; 16(2): 292â311. Publisher Full Text\n\nCain DS:Social work practice in Disasters.Corcoran K, Roberts AR, editors. Social Workersâ Desk Reference. 3rd ed.Oxford University Press;2015; pp. 130â139.\n\nCarroll B, Balogh R, Morbey H, et al.: Health and social impacts of a flood disaster: responding to needs and implications for practice. Disasters. 2010; 34(4): 1045â1063. PubMed Abstract | Publisher Full Text\n\nComas-Herrera A, Fernandez JS, Hancock R, et al.: COVID-19: Implications for the Support of People with Social Care Needs in England. J. Aging Soc. Policy. 2020; 32(4-5): 365â372. PubMed Abstract | Publisher Full Text\n\nDaly M: COVID-19 and care homes in England: What happened and why? Soc. Policy Adm. 2020; 54(7): 985â998. PubMed Abstract | Publisher Full Text\n\nde Jonge E , Kloppenburg R, Hendriks P: The impact of the COVID-19 pandemic on social work education and practice in the Netherlands. Soc. Work. Educ. 2020; 39(8): 1027â1036. Publisher Full Text\n\nDekel R, Baum N: Intervention in a Shared Traumatic Reality: A New Challenge for Social Workers. Br. J. Soc. Work. 2010; 40(6): 1927â1944. Publisher Full Text\n\nDevlieghere J, Roose R: Social work during the Covid-19 pandemic: staying close while maintaining social distancing. Eur. J. Soc. Work. 2020; 23(4): 541â542. Publisher Full Text\n\nDrummond DA, Carey LB: Chaplaincy and Spiritual Care Response to COVID-19: An Australian Case Study â The McKellar Centre. Health Soc. Care Chaplain. 2020; 8(2): 165â179. Publisher Full Text\n\nFabre C: Social Rights under the Constitution. Government and the Decent Life. Oxford University Press;2004.\n\nFallon A, Dukelow T, Kennelly SP, et al.: COVID-19 in nursing homes. QJM: Int. J. Med. 2020; 113(6): 391â392. PubMed Abstract | Publisher Full Text\n\nFischer F, Raiber L, Boscher C, et al.: COVID-19 and the Elderly: Who Cares? Front. Public Health. 2020; 8(151). PubMed Abstract | Publisher Full Text\n\nGarbers Y, Konradt U: The effect of financial incentives on performance: A quantitative review of individual and team-based financial incentives. J. Occup. Organ. Psychol. 2014; 87: 102â137. Publisher Full Text\n\nKhatri J, Fitzgerald G, Poudyal Chhetri MB: Health risks in disaster responders: a conceptual framework. Prehosp. Disaster Med. 2019; 34(2): 209â216. PubMed Abstract | Publisher Full Text\n\nKuru N, Kublay G: The effect of laughter therapy on the quality of life of nursing home residents. J. Clin. Nurs. 2017; 26(21-22): 3354â3362. PubMed Abstract | Publisher Full Text\n\nKoenig HG: Ways of Protecting Religious Older Adults from the Consequences of COVID-19. Am. J. Geriatr. Psychiatry. 2020; 28(7): 776â779. PubMed Abstract | Publisher Full Text\n\nLandry AT, Gagné M, Forest J, et al.: The Relation Between Financial Incentives, Motivation, and Performance. J. Pers. Psychol. 2017; 16(2): 61â76. Publisher Full Text\n\nLindberg DA: Integrative Review of Research Related to Meditation, Spirituality, and the Elderly. Geriatr. Nurs. 2005; 26(6): 372â377. PubMed Abstract | Publisher Full Text\n\nMedina A: Promoting a culture of disaster preparedness. J. Bus. Contin. Emer. Plan. 2016; 9(3): 281â290. PubMed Abstract\n\nMinistry of Family and Social Policy: Sprawozdanie MRPiPS-05 za 2020 rok o placówkach zapewniajÄ
cych caÅodobowÄ
opiekÄ i wsparcie.August 2022.Reference Source\n\nNecel R: The Coronavirus Pandemic in Nursing Homes â the Experience of Care Workers in Poland. [Dataset].2022, August 29. Publisher Full Text\n\nNewell JM, Nelson-Gardell D: A Competency-Based Approach to Teaching Professional Self-Care: An Ethical Consideration for Social Work Educators. J. Soc. Work. Educ. 2014; 50(3): 427â439. Publisher Full Text\n\nNiessen L, Grijseels E, Rutten F: The evidence-based approach in health policy and health care delivery. Soc. Sci. Med. 2000; 51(6): 859â869. Publisher Full Text\n\nNilsson E, Olaison A: Needs assessment in social work with older people in times of Covid-19: Initial ideas from an empirical study. J. Soc. Work. 2020; 4(2): 52â60. Publisher Full Text\n\nRaine R: Evidence-based policy: rhetoric and reality. J. Health Serv. Res. Policy. 1998; 3(4): 251â253. PubMed Abstract | Publisher Full Text\n\nRevet S, Langumier J: Governing Disasters. Beyond Risk Culture. Palgrave Macmillan;2015.\n\nRevet S: Disasterland. An Ethnography of the International Disaster Community. Palgrave Macmillan;2020.\n\nRodrigues R, Simmons C, Schmidt AE, et al.: Care in times of COVID-19: the impact of the pandemic on informal caregiving in Austria. Eur. J. Ageing. 2021; 18: 195â205. PubMed Abstract | Publisher Full Text\n\nRothman J: Multi Modes of Intervention at the Macro Level. J. Community Pract. 2007; 15(4): 11â40. Publisher Full Text\n\nScheyett A: Thoughts in the Time of COVID-19. Soc. Work. 2020; 65(3): 209â211. PubMed Abstract | Publisher Full Text\n\nShue H: Basic Rights. Subsistence, Affluence, and U.S. Foreign Policy. Princeton University Press;1980.\n\nSkinner J:Social Work Practice and Personal Self-care.Corcoran K, Roberts AR, editors. Social Workersâ Desk Reference. 3rd ed.Oxford University Press;2015; pp. 130â139.\n\nStatistics Poland: ZakÅady stacjonarne pomocy spoÅecznej w 2020 r.May 2021.Reference Source\n\nSwift C: Being There, Virtually Being There, Being Absent: Chaplaincy in Social Care During the COVID-19 Pandemic. Health Soc. Care Chaplain. 2020; 8(2): 154â164. Publisher Full Text\n\nSwinford E, Galucia N, Morrow-Howell N: Applying Gerontological Social Work Perspectives to the Coronavirus Pandemic. J. Gerontol. Soc. Work. 2020; 63(6-7): 513â523. PubMed Abstract | Publisher Full Text\n\nWeicht B: The Meaning of Care. The Social Construction of Care for Elderly People. Palgrave Macmillan;2015.\n\nWenger E: Communities of Practice: Learning, Meaning, and Identity. Learning in Doing: Social, Cognitive and Computational Perspectives. Cambridge University Press;1998."
}
|
[
{
"id": "149759",
"date": "25 Oct 2022",
"name": "Ewa Kantowicz",
"expertise": [
"Reviewer Expertise Social work theory",
"methodology of sw research",
"child protection",
"family support",
"profssionalization of social work",
"comparative social work."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. Evaluation of the research background\nThe conducted empirical research was to partially fill the gap in knowledge about the condition of care workers in Poland during the coronavirus pandemic. The research was based on the culture-of-disaster perspective presented e.g. by Sandrine Revetand and Julien Laugumier, which focuses on âwhat disaster is made of for each of the actors involvedâ (Revet & Langumier, 2015).\nThe subject of interest was focused on the attitudes toward the crisis situation shared by individuals, including how they experienced the reality of the pandemic. The proposed cultural approach, the experiences of care workers were considered as the important ones, because they were treated as a social mirror of the resourcefulness of institutions in the crisis caused by the pandemic. The issues raised undoubtedly determine the importance of the issue raised in the context of resident care for dependent persons, but also the use of knowledge and experience from the pandemic period of employees of these institutions in securing adequate medical, psychological and social care and a support system corresponding to the needs of the wards.\n2. Evaluation of methodology\nThe field research was carried out in April 2021. It was conducted using the computer-assisted web interviewing (CAWI) technique. The CAWI questionnaire consisted of 19 questions grouped in following topics: mental well-being of employees, internal rules of the functioning of the institution, residentsâ needs, cooperation with the environment, birth certificate questions. The CAWI technique was chosen because it provided easy access to the population, which was particularly important during the pandemic due to the need to maintain social distancing. This tool allowed also the avoidance of open-ended questions and questions formulated primarily in the form of statements, making it useful in this project. The statistical analyses conducted (chi-square test or Fisherâs exact test) showed that the assessments of the functioning of nursing homes during the pandemic were different depending on the respondentsâ job position.\nIn the course of the analyses, the hypothesis has been confirmed that the type of nursing home in which an employee works significantly differentiates the assessment of the functioning of the care institution in the context of meeting the basic needs of residents. It was interesting that the surveyed nursing home employees most often indicated the necessity to work under the conditions of health risk as a stressful factor. Another stressful factor for respondents was their responsibility for the residents of nursing homes. The analyses conducted (chi-square test and Fisherâs exact test) had confirmed the hypothesis that opinions about stressors differ depending on seniority. Statistically significant differences were noted in the opinions on combining work with responsibilities toward loved ones. The analyses also confirmed the hypothesis about the relationship between the position held and the importance given to unclear procedures at the local level. The research results indicated the widespread experience of increased stress among nursing home employees. The analysis of statistical correlations performed using the chi-square test did not show significant differences in the opinions on the forms of psychological support provided depending on the position held, seniority, the type of nursing home and the type of municipality in which the facility was situated. In the research, stressors had been identified and changes in the mental wellbeing of nursing home workers during the pandemic period.\nThe research methodology was used correctly and allowed to formulate some proposals for social practice. The analysis of the data allowed to design and implement forms of support most suitable for the reported needs, based on the direct experiences of care workers. The organization of various forms of mental health support for formal care workers, including nursing staff, is the main challenge facing the social welfare system in Poland.\n3. General evaluation of the content of article\nThe main objective of this article was to analyze the attitudes of care workers in nursing homes toward the reality of the pandemic in Poland, in four basic contexts: assessment of the degree of social care in the pandemic era; assessment of the most stressful factors since the outbreak of the pandemic; assessment of the mental wellbeing of care workers and evaluation of institutional measures taken to improve mental health. The issues raised are a very important topic, which is e.g. the results of the experience of the pandemic in recent years, as well as current problems related to the protection of people under residential care.\n4. Formal evaluation\nThe article is factually and methodologically correct and fits into the current discourse in the area of social work. The layout of the article is appropriate and structurally corresponds to articles presenting the results of empirical research. The applied terminology is part of research in the field of social sciences, with a particular focus on social work. The applied methodology of quantitative research, as well as the criteria for selecting a research sample correspond to the specificity of this type of research, creating space for their thematic and statistic interpretation and final reflection of results. The literature taking into account newer proposals in this field was used properly and allowed for the confrontation of the results with other studies in the discussion and summary.\nFinal remarks\nThe conducted empirical research was to partially fill the gap in knowledge about the condition of care workers in Poland during the coronavirus pandemic. The proposed cultural approach, the experiences of care workers were considered as the important ones, because they were treated as a social mirror of the resourcefulness of institutions in the crisis caused by the pandemic. The issues raised from the research undoubtedly determine the importance of the problems raised in the context of resident care for dependent persons, but also the use of knowledge and experience from the pandemic period of employees of these institutions in securing adequate medical, psychological and social care and a support system corresponding to the needs of the wards. The research methodology was used correctly and allowed to formulate some proposals for social practice. The analysis of the data allowed to design and implement forms of support most suitable for the reported needs, based on the direct experiences of care workers. The organization of various forms of mental health support for formal care workers, including nursing staff, is the main challenge facing the social welfare system in Poland.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "154261",
"date": "03 Jan 2023",
"name": "Florin Lazar",
"expertise": [
"Reviewer Expertise Social work",
"Social policy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is generally well structured and written. The main topic is relevant for policy-making and brings into the debate about the impact of the Covid-19 pandemic the perspective of care workers in nursing homes. The experiences from the Polish respondents could be relevant to other contexts as well, but the manuscript needs some revisions before it is approved.\nHere are some comments and suggestions for improvements:\nThe first sentence needs a âspaceâ in the word âofallâ to read âNursing homes constitute the largest number of all care institutions in Poland. â\nThe reference to Statistics Poland need to be translated into English also (in the Reference section).\nIs not clear why the author consider that data on morbidity of medical and care staff during the pandemic explains the impact of the pandemic on the care system. Also, the phrasing âcapacity and effectiveness of the care systemâ is not clear enough as it does not explain the type of capacity (capacity to do what?), nor what is meant by effectiveness of the care system (presumably to protect the service users from the negative consequences of the pandemic - from this point of view, maybe data on morbidity of residents could be more relevant). The next idea is not linked to the previous phrase, as the expansion of evidence-based health services does not explain the insufficient data about the care system in Poland. Moreover the argument is that evidence-based research developed over the past 30 years on the âhealth of the populationâ, but the focus of the current research is on the perceptions of care workers and less on their health (apart from mental health). The title is a bit misleading as the research is focused on care workers in nursing homes and not all care workers (e.g. in community-based services, foster care, etc.).\nChange âBackgroundsâ to âBackgroundâ.\nThe âculture-of-disasterâ approach is not referred back in the Discussion section, but only presented in the Introductory section. It would have been relevant to discuss to what extent the data collected confirmed the initial theoretical perspective.\nThe Background section is missing information about the situation of nursing homes in Poland and why were these institutions were chosen. Some information is provided later on, but I suggest to move the text somewhere in the Background section.\nThe objective and hypotheses are not clear enough, as is stated that assessments are made, but is not sufficiently explained how these assessments are made. These are rather perceptions or attitudes, not really assessments. For instance âassessment of the degree of social careâ is not clear, nor it is operationalised to understand what is meant my degree of social care, so I would suggest to avoid using the terms âassessmentsâ and âhypothesesâ, but rather âattitudesâ or âperceptionsâ and âresearch questionsâ. The alternative is to provide sufficient details about how the assessment and the concepts from the hypotheses are measured. A quantitative research does not always include hypotheses, if it is descriptive (see Rubin, A., & Babbie, E. R. (2016). Empowerment series: Research methods for social work. Cengage Learning.).\nThe urban-rural option for residence is unusual and the author need to explain how a place is both urban and rural?\nThe current hypotheses are not clear-cut, being too general and comprising too many variables to test. If kept, I suggest to choose a more precise statement - e.g. managers are more likely to be worried about the general impact of the pandemic on the institution than frontline workers. Then every concept is detailed on how it is measured. Currently no information is available on how the concepts are measured - e.g. degree of social care, stressful factors, mental wellbeing, institutional measures. The reader find out only in the Results section something about these. Irrespective if the author decides to keep the hypotheses or not, these changes are needed.\nThe survey is presented as an interview (CAWI), so I suggest to adjust the text to reflect the situation. From the questionnaire presented as Extended data, it seems that Microsoft Forms from the software Microsoft 365 package was used.\nRegarding Ethical approval, it seems that it was obtained after data collection and not before as it usually is needed. However, the data collection process did not pose ethical concerns, as participation was voluntarily.\nThe reference to â(Ministry of Family and Social Policy, 2022)â from the second sentence of the Results section need to be moved after the first sentence and not after the second sentence, since it gives information about the target population and not about the number of those included in the research. However, some info about the coverage of the study participants would be necessary. In the five provinces included in the research there were about 262 nursing homes (54 nursing homes in Lower Silesia, 55 in the Lodz Province, 47 in the Subcarpathian Province, 44 in the Lublin Province and 62 in the Greater Poland Province). Since the number of respondents was 189, we can assume there were responses from about 95 institutions, out of 262 (about 36% coverage rate; another option is to report the coverage rate based on the total number of all employees in nursing homes in the five provinces - about 1%).\nI suggest to add a table describing the study population in the Results section.\nRegarding the presentation of the results, I suggest to present the tables with the correlations (including significance coefficients) instead of the frequencies. Also, I suggest to either group negative vs. positive responses, or calculate means and present the results referring to the hypotheses, if kept.\nIn the sub-section âAssessment of mental healthâ the whole first paragraph could be moved into a new sub-section within Methods section, âMeasuresâ and add info about the other measures used (e.g. in the next sub-section, the first 4 sentences + the paragraph about the expected support could be also moved).\nThe Discussion section could start with a summary of the main findings and then point out when is referring to own findings and when to those arising from the literature.\nI suggest to add a limitation of the research related to the conflict of interest of respondents assessing their work, presumably being more likely to be positive.\nThe suggestion to provide âlaughter therapy (Kuru & Kublay, 2017) or meditation-relaxation strategies (Lindberg, 2005),â does not derive from the research itself, so I suggest to remove it or to add something more general, as I assume there are other forms of support available. I also suggest to end with a more general conclusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9226",
"date": "14 Feb 2023",
"name": "Ryszard Necel",
"role": "Author Response",
"response": "The first sentence needs a âspaceâ in the word âof allâ to read âNursing homes constitute the largest number of all care institutions in Poland. The sentence is corrected - p. 2 The reference to Statistics Poland need to be translated into English also (in the Reference section). The reference is corrected - p. 21 Is not clear why the author consider that data on morbidity of medical and care staff during the pandemic explains the impact of the pandemic on the care system. The text has been added - p. 2-3 the argument is that evidence-based research developed over the past 30 years on the âhealth of the populationâ, but the focus of the current research is on the perceptions of care workers and less on their health (apart from mental health) The sentence is corrected - p. 3 The title is a bit misleading as the research is focused on care workers in nursing homes and not all care workers (e.g. in community-based services, foster care, etc.). In the first sentence of the title it indicates nursing homes Change âBackgroundsâ to âBackgroundâ. The sentence is corrected - p. 3 The âculture-of-disasterâ approach is not referred back in the Discussion section, but only presented in the Introductory section. It would have been relevant to discuss to what extent the data collected confirmed the initial theoretical perspective. The text has been added â p. 15 The Background section is missing information about the situation of nursing homes in Poland and why were these institutions were chosen. Some information is provided later on, but I suggest to move the text somewhere in the Background section. The text has been added â p. 3 The objective and hypotheses are not clear enough, as is stated that assessments are made, but is not sufficiently explained how these assessments are made. These are rather perceptions or attitudes, not really assessments. For instance âassessment of the degree of social careâ is not clear, nor it is operationalised to understand what is meant my degree of social care, so I would suggest to avoid using the terms âassessmentsâ and âhypothesesâ, but rather âattitudesâ or âperceptionsâ and âresearch questionsâ. The alternative is to provide sufficient details about how the assessment and the concepts from the hypotheses are measured. A quantitative research does not always include hypotheses, if it is descriptive (see Rubin, A., & Babbie, E. R. (2016). Empowerment series: Research methods for social work. Cengage Learning.). 1) As suggested by the second reviewer. I removed hypotheses and replaced them with research questions  - p. 3 I have seen Rubin, A., & Babbie, E. R. (2016). Empowerment series: Research methods for social work. Cengage Learning. Thank you for this book. 2) The section âDescription of variablesâ has been added. I have presented in the table type of variables and items. Now I hope is clearer what is meant the main variables â p.7-8 3) I also removed the term âassessmentâ and replaced it with âperceptionâ. Regarding the presentation of the results, I suggest to present the tables with the correlations (including significance coefficients) instead of the frequencies. Also, I suggest to either group negative vs. positive responses, or calculate means and present the results referring to the hypotheses, if kept. The analysis of each variable was separately (seniority, type of commune, etc.). Then it would be necessary to present several tables in a short manuscript. some info about the coverage of the study participants would be necessary. The text has been added â p. 8 I suggest to add a table describing the study population in the Results section. The table has been added â p. 8-9 The urban-rural option for residence is unusual and the author need to explain how a place is both urban and rural? The text has been added â p. 3 The Discussion section could start with a summary of the main findings and then point out when is referring to own findings and when to those arising from the literature. The text has been added â p. 15 I suggest to add a limitation of the research related to the conflict of interest of respondents assessing their work, presumably being more likely to be positive. The text has been added â p. 17 The suggestion to provide âlaughter therapy (Kuru & Kublay, 2017) or meditation-relaxation strategies (Lindberg, 2005),â does not derive from the research itself, so I suggest to remove it or to add something more general, as I assume there are other forms of support available. I also suggest to end with a more general conclusion The text has been removed I also suggest to end with a more general conclusion The text has been added â p. 18"
}
]
}
] | 1
|
https://f1000research.com/articles/11-1008
|
https://f1000research.com/articles/12-317/v1
|
22 Mar 23
|
{
"type": "Research Article",
"title": "Restinga ectomycorrhizae: a work in progress",
"authors": [
"Ariadne N. M. Furtado",
"Marco Leonardi",
"Ornella Comandini",
"Maria Alice Neves",
"Andrea C. Rinaldi",
"Marco Leonardi",
"Ornella Comandini",
"Maria Alice Neves",
"Andrea C. Rinaldi"
],
"abstract": "Background: The Brazilian Atlantic Forest is one of the most biodiverse terrestrial ecoregions of the world. Among its constituents, restinga vegetation makes a particular case, acting as a buffer zone between the oceans and the forest. Covering some 80% of Brazilian coastline (over 7,300 km in length), restinga is a harsh environment where plants and fungi interact in complex ways that just now are beginning to be unveiled. Ectomycorrhizal symbiosis, in particular, plays a so far ungauged and likely underestimated role. We recently described the morpho-anatomical and molecular features of the ectomycorrhizae formed by several basidiomycetous mycobionts on the host plant Guapira opposita, but the mycorrhizal biology of restinga is still largely unexplored. Here, we report new data on the ectomycorrhizal fungal symbionts of G. opposita, based on the collection of sporomata and ectomycorrhizal root tips in restinga stands occurring in southern Brazil. Methods: To obtain a broader view of restinga mycorrhizal and ecological potential, we compiled a comprehensive and up-to-date checklist of fungal species reported or supposed to establish ectomycorrhizae on restinga-inhabiting host plants, mainly on the basis of field observations. Results: Our list comprises some 726 records, 74 of which correspond to putative ectomycorrhizal taxa specifically associated with restinga. These include several members of Boletaceae, Amanita, Tomentella/Thelephora, Russula/Lactifluus, and Clavulina, as well as hypogeous fungi, like the recently described Longistriata flava. Conclusions: Our survey reveals a significant diversity of the restinga ectomycorrhizal mycobiota, indicating the importance of this symbiosis for the ecological functioning of a unique yet poorly known and threatened ecosystem.",
"keywords": [
"Atlantic Forest",
"Brazilian fungi",
"diversity",
"ectomycorrhiza",
"fungal conservation",
"mycorrhizal symbiosis",
"Neotropics"
],
"content": "Introduction\n\nUnderstanding how communities come together has been a primary goal of researchers over the last century. In addition to the diversity of organisms and their multiple strategies to resist environmental conditions, interspecific interactions add another layer of complexity to the structure of communities.1 The mycorrhizal symbiosis is one of the most prominent and ecologically crucial mutualistic associations found in terrestrial habitats.2 Plant and fungal partners interact in the rhizosphere, which contributes significantly to nutrient cycling and carbon sequestration.3,4 The composition of mycorrhizal fungi in an ecosystem directly affects plant community structure, and environmental factors that influence species diversity over time have an impact on host selectivity for plant communities and fungal associations.5\n\nResearch on arbuscular mycorrhizae (AM) is quite advanced in Brazil, with over 40 years of history.6 However, our fascination with plant-fungus interactions took a different path allowing us to focus and investigate another intriguing mutualistic association, the ectomycorrhizae (ECM), from natural habitats of the Brazilian Atlantic Forest, particularly from the restinga. At the global scale, ectomycorrhizal plants have been documented in approximately 335 genera and 8,500 species, with recent research indicating that a large portion of plant symbionts have yet to be confirmed, specifically in the tropics.7 On the other hand, ectomycorrhizal fungi have been assigned to 236 genera and approximately 20,000-25,000 species,8 which is a small number when compared to current estimates of 2.2 to 3.8 million fungal species diversity.9\n\nIn Brazil, ectomycorrhizal fungi often have a fragmented distribution due to the lack of information about them and because they do not always have the same distribution as the host plants.10,11 This can result in high endemism at the species level due to the specific habitats they occupy.12â14 In 2016, Roy and coworkers reported approximately 180 species of ectomycorrhizal fungi in Brazilian native forests.15 In fact, the majority of the published studies were conducted in introduced Pinus and Eucalyptus plantations.14,16â18 As a result, the diversity of ectomycorrhizal fungi associated with native plants of the Atlantic Forest still needs understanding.14,15,19\n\nThe Atlantic Forest, which is home to several endemic species, is one of the world's top 25 priority areas for biodiversity conservation.20 The high diversity of potentially ectomycorrhizal plant species suggests a large but still unknown diversity of ectomycorrhizal partners.11 These relationships can be generalist,21 however, there are cases of proven specificity, resulting from a long process of joint evolution between plants and fungi.5 The Atlantic Forest includes ecosystems such as the restinga, where at least 700 specimens of typically ectomycorrhizal fungal taxa have already been collected.22\n\nThe restinga was one of the first environments to be harmed by human intervention. Currently, 79% of the Brazilian coast is covered by restinga, representing an essential constituent of the Atlantic Forest.23 However, in terms of biodiversity and conservation status, these ecosystems remain poorly understood. Restinga vegetation is associated with Quaternary coastal sand deposits and rocky coastal habitats,24 grows on sandy soils near the sea between lake formations and/or dunes and, as it moves away from the ocean, is composed of creeping plants, shrubs and trees, including forming forests.25\n\nDue to the high frequency of symbiotic interaction in the restinga, ECM have a potentially critical role in restoration and management interventions in these ecosystems.26 ECM can improve host plant resistance to pathogens through direct competition;27 they can increase plant drought tolerance by improving plant-soil contact surface, host plant water conductivity, and resistance to high soil salinity by restricting sodium uptake by plant tissues and activating stress response pathways.28 Sadly, anthropic activities are negatively affecting the diversity and functionality of the ectomycorrhizal community in forest soils due to soil erosion, changes in land use, inorganic toxins, fire, and non-native plant invasions.29 Such processes have promoted the elimination of many populations and, potentially, the decrease of the genetic diversity of several species.30 Nevertheless, these ecosystems are not considered priority conservation areas, and the high degree of degradation observed becomes especially harmful in a scenario of accelerated climate change.31 Studies that aim to understand ectomycorrhizal interactions of restinga are important to help develop conservation and restauration projects. However, they are still in their early stages, especially because the majority of them are based solely on the presence of sporomata. To date, only two works have been published that link both fungus and plant partners in the restinga and present a detailed morphological and molecular characterization of the ECM (Hysterangium atlanticum + Coccoloba spp.32 and Amanita viscidolutea + Guapira opposita33). Researches that have been developed in Brazil corroborate the urgent need to better understand the belowground diversity in the Atlantic Forest, especially considering that the restinga potentially harbors a unique community of mycorrhizal taxa.34 In this work we present new information on ECM from restinga, through collections of basidiomata, as well as ectomycorrhizal root tips. Furthermore, based on survey data from national fungaria and herbaria, published literature, field observations, and molecular approaches, we provide a comprehensive and updated list of fungal species reported as (or supposed to) establishing ECM with native plants in the restinga. The data presented here reveal a high diversity of ectomycorrhizal fungi in the restinga and are discussed further below.\n\n\nMethods\n\nThe restinga consists of a transition zone (ecotone) that acts as a buffer zone between the oceans and the forests and includes Holocene sands of marine origin.25 Because of rapid leaching and the closeness of the ocean, the soil is nutrient-poor and water deficient, with high pH and it is highly salinized.35 The community of ectomycorrhizal fungi, as well as plant symbionts, are structured and maintained in part by all of these factors.19 Its vegetation is geologically young and originates from other ecosystems (Atlantic Forest, Amazon, Cerrado and Caatinga), but it exhibits phenotypic variation when compared to the habitat of origin, making it a unique, extreme ecosystem that requires specific adaptations and a high level of ecological plasticity.36 As a result, the restinga diversity pattern varies greatly across its geographic range.37\n\nRestinga cover approximately 80% of the Brazilian coast, the equivalent to 7,360 Km in length, spanning all coastal states (Figure 1).23 In addition to providing habitat and refuge for many species for at least part of their life cycles, the restinga stores rainwater and assists in flood control and water cycle regulation.25\n\nCollections were made between October 2017 and May 2019 in three restinga areas in Florianópolis, Brazil: Parque Natural Municipal das Dunas da Lagoa da Conceição (-27.694028, -48.506587), Monumento Natural Municipal da Lagoa do Peri (-27.728243, -48.510175) and Parque Natural Municipal da Lagoa do Jacaré das Dunas do Santinho (-27.467783, -48.393395). The basidiomata were photographed in the field and identified by comparing them with the morphology described in the literature and by using DNA barcoding of the ITS region. Fungal species names were inspected in Index Fungorum (RRID:SCR_008975) and MycoBank (RRID:SCR_004950) for nomenclatural and taxonomic synonyms, and current names were adopted. After making morpho-anatomical analyses, the basidiomata were dried at 40 °C for further preservation. The identification of the plant was made by a botanist and confirmed by sequence similarity of the ITS region [38 Caddah personal communication].\n\nFor ECM, soil samples (approximately 20 cm3) were collected below the basidiomata and left in water overnight. The roots were washed and carefully selected under a stereomicroscope. The roots that had an ectomycorrhizal mantle were morphotyped following the standard methodology and terminology used for studying ECM.39 From each root system with a mantle, several tips were transferred to 70% alcohol and stored at -20 °C for subsequent DNA extraction. Also, part of the root system with the mantle was fixed in 4% glutaraldehyde for morpho-anatomical analyses. Voucher material of the basidiomata, ECM and the host plant are deposited in the FLOR herbarium and fungarium and the permanent collection of the mycology laboratory (Micolab) at the Universidade Federal de Santa Catarina (UFSC), Florianópolis, Brazil.\n\nGenomic DNA was extracted from the basidiomata using a PowerPlant®Pro DNA Isolation Kit (MO BIO Laboratories, Inc.), following the manufacturer's protocol adapted for fungi. The internal transcribed spacer of ribosomal nuclear DNA (nrITS) region was amplified using the primers ITS1F and ITS440 and the following cycling parameters: an initial denaturation at 94 °C for 2 min; 40 cycles of 30 s at 94 °C, 45 s at 55 °C and 1 min at 72 °C; and a final extension at 72 °C for 7 min. A direct PCR approach was applied to amplify the ITS region from the ECM tips isolated from soil samples41 using the same pair of primers (ITS1F/ITS4). A total of 2 ml of 20 mg/ml Bovine Serum Albumin (BSA) solution was added to each reaction tube to prevent PCR inhibition. The parameters applied to the PCR cycles followed Leonardi et al.42 To identify the host plant, plant DNA was extracted from the ECM root tips using an isolation kit (see above), and the ITS region for the plant was amplified using the ITS-u1 and ITS-u4 primer pair38 and the following cycle parameters: an initial denaturation at 94 °C for 4 min; 34 cycles of 30 s at 94 °C, 40 s at 55 °C and 1 min at 72 °C; and a final extension at 72 °C for 10 min. The DNA extracted from Guapira opposita leaves was used as a positive control. Sanger sequencing was performed with a BigDye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, California, USA) at the company Myleus Biotecnologia, in Minas Gerais, Brazil, following the manufacturerâs instructions and using the same primers cited above for the correspondent symbionts. New sequences generated during this work were included in GenBank43 and the accession numbers for the sequences are shown in Table 1.\n\nReport of naturally occurring ectomycorrhizal fungi, potential host and/or sequence isolated from basidiomata or roots. All other records are about basidiomata collections, if not specified otherwise. Asterisk (*) refers to potential hosts which have not been confirmed. For names of fungal taxa and synonymy, we followed Index Fungorum (http://www.indexfungorum.org/) and MycoBank (http://www.mycobank.org).\n\nThe data provided here on the relationship between restinga plants and ectomycorrhizal fungi are mainly based on field observations reports. Personal observations and information collected from a wide range of published and platform sources are included in the data source. The Brazilian fungaria collections (through SpeciesLink network) and available literature databases (e.g., Scopus (RRID:SCR 022559), PubMed (RRID:SCR 004846), ISI Web of Science (RRID:SCR 022706), ResearchGate (RRID:SCR 006505)) were searched for records on potential restinga host plants and related mycobionts. Only species belonging to fungal genera for which the ectomycorrhizal status has been proved or is considered likely were considered for listing.8,11,44 Listed sequences of restinga ECM fungi (Table 1) are those reported in relevant publications and were retrieved from either GenBank or UNITE. Some of the punctual ectomycorrhizal records were based only on the presence of the sporome next to a known plant symbiont, without any direct confirmation of the presence of ectomycorrhiza. As a result, these data are susceptible to non-measurable errors, particularly when more than one potentially ectomycorrhizal plant is in the environment. Despite our efforts to scan as many bibliographic sources as possible, our survey may be partial and incomplete.\n\n\nResults and Discussion\n\nRolf Singer, a true pioneer in the study of mycorrhiza biology in South America, once wrote that he believed three âectotrophic regionsâ did exist in the continent, each one characterized by a single host plant genus. In a seminal work for the field, he listed the Quercus humboldtii area in Colombia, the ecosystem formed by Alnus jorullensis in the Andes and, more extensively, the Nothofagus region in Chile and Argentina.45 Beyond these areas, Singer stated, ectomycorrhizal symbiosis in South America was restricted to plantations of imported trees, in particular Pinus.46 However, as his knowledge of various types of forests in temperate and tropical South America improved, Singer expanded his view. âOur own recent investigation in the Lower Rio Negro region of Central Amazonia show, that certain vegetation types (campina, campinarana, igapó) are rich in ectomycorrhiza-forming fungi, e.g., Boletaceae ⊠Thus, in both hemispheres, certain tropical soils require for the formation of any kind of forest the presence of ectomycorrhiza,â Singer remarked in 1979.47,48\n\nSince Singerâs times, our understanding of the distribution, relevance and role of ectomycorrhizal symbiosis in many ecological settings in temperate, tropical and subtropical South America has grown considerably, but not so rapidly as one could have expected given the premises. Indeed, while Singer and colleagues just supposed the ectomycorrhizal status of many fungal species and relevant host plants on the basis of field observations, detailed studies able to identify and describe fungal structures on the roots of ectotrophic plants in most South American ecosystems began only in the last decade of the twentieth century, i.e., considerably later than in the Northern Hemisphere.49 According to the recent account of the currently known biogeographic pattern of ectomycorrhizal symbiosis in South America by Nouhra and colleagues,49 three main regions can be recognized, broadly confirming Singerâs vision but also elaborating on it: 1) the Northern Andean cordillera, with mostly temperate forests, where ECM such as Quercus, Colombobalanus, Alnus and Salix occur; 2) the sub Antarctic forests in far Southern America, dominated by ectomycorrhizal trees in the Nothofagaceae (Fuscospora, Lophozonia and Nothofagus); the Guiana Shield region and the coastal vegetation of the Atlantic rainforests of Brazil, where a large (and fast growing) number of ectomycorrhizal fungi in the/cortinarius, /russula-lactarius, /amanita and/clavulina lineages have been spotted in recent times with their associated host plants, including Dicymbe, Aldina (Fabaceae), Pseudomonotes (Dipterocarpaceae), Pakaraimaea (Cistaceae), Coccoloba (Polygonaceae), Gnetum (Gnetaceae), Pisonia, Neea, and Guapira (Nyctaginaceae). And is here that our story becomes more specific and very personal, as outlined below.\n\nMycorrhizal symbiosis plays a crucial role in basically each and every terrestrial ecosystem,3 and restinga are not an exception. For many years, however, this peculiar coastal habitat has been the object of studies delving exclusively into the communities of arbuscular mycorrhizal fungi and their plant relationships, while almost no attention whatsoever has been devoted to the ectomycorrhizal component. We thus started investigating the spread, diversity and ecology of the ECM-fungal contingent, not only by recording the occurrence of sporomata of supposedly ectomycorrhizal macrofungi, but also looking directly at the roots and the structures therein. What we found was surprising, indeed. Working mainly at a restinga in the Parque Natural Municipal das Dunas da Lagoa da Conceição in Florianópolis, Brazil, we rapidly understood that Guapira opposita (Vell.) Reitz. is a hub for local ectomycorrhizal community, hosting a range of fungal species on its roots. Out of a total of 29 morphotypes collected from soil samples, 10 were found associated with G. opposita roots, all corresponding to Basidiomycota taxa, based on molecular barcoding.50 The best represented clade was/tomentella-thelephora, with Tomentella bursting six species (Table 1); of note, two macrofungi native species from the restinga of the Atlantic Forest, namely Amanita viscidolutea and Austroboletus festivus, besides occurring as basidiomata were also found associated to G. opposita roots in our survey (Table 1).50 The most striking characteristics of Guapira ECMs, however, remain with their morpho-anatomical features, that make them rather unique. The short, simple or long, thin branched ectomycorrhizal systems, close connections between the layered mantle and the cortical cells, absence of a Hartig net or other fungal elements in the cortex are diagnostic characteristics that make the Guapira ECMs we observed rather unique, to the point that we proposed the term âGuapirioidâ to distinguish them from the other known ectomycorrhizal types (Figure 2).33,50 Our study on the ectomycorrhiza of A. viscidolutea on G. opposita has been the first detailed morpho-anatomical and molecular characterization of a naturally occurring mycorrhiza associated with a native plant host in restinga forest in South America.33 Besides G. opposita, we can also find members of the following potential ectomycorrhizal families growing in restinga: Fabaceae, Moraceae, Myrtaceae, Nyctaginaceae, Polygonaceae, and Salicaceae.51\n\n(a) Amanita viscidolutea; (b) Austroboletus festivus; (c) Inocybe sp.; (d) Thelephora sp1.; (e) Tomentella sp1. and Tomentella sp2.; (f) Tomentella sp3.; (g) Tomentella sp4.; (h) Tomentella sp5.; (i) Tomentella sp6.\n\nAs mentioned above, representatives of/tomentella-thelephora clade were the most frequently encountered taxa in our restinga surveys, a finding in line with studies based on root and soil analysis that revealed that the/tomentellaâthelephora clade is diverse and dominant in neotropical habitats,52,53 although they are generally undersampled due to their inconspicuous basidiomata, which makes it difficult to identify the sampled taxon. It is known that members of Nyctaginaceae establish ectomycorrhizal associations with a low ECM fungal diversity in the Neotropics.19,54 The mycorrhizal status of the family seems to be not homogeneous, with several species confirmed as dual-mycorrhizal55,56 and many others believed to be non-mycorrhizal.7,57 Moreover, some of the mycorrhizal structures observed in Nyctaginaceae escape classical classification. In the so-called âPisonioidâ mycorrhizae of Pisonia, Hartig net is poorly or not developed, and instead âtransfer cellsâ are observable in the epidermis and cortex of the host root.58,59 Haug et al.,60 and Ãlvarez-Manjarrez et al.,61 observed Tomentella/Thelephora and Membranomyces ectomycorrhizae associated with Guapira roots in Ecuador and Mexico, respectively. In both cases, they pointed to the presence of intraradical hyphae in the roots and the possibility of Guapira species forming a type of ectendomycorrhiza. On the basis of their work on the ectomycorrhizal types of Nyctaginaceae genera Neea and Pisonia in South Ecuador, Haug and colleagues54 concluded that the set of observed characters (the combination of long root systems that are only partly transformed into ectomycorrhizae, with root hair formation that is not suppressed, occasional intracellular penetration of hyphae, and sporadic formation of transfer cell-like structures) may suggest that Nyctaginaceae represent an early step in the evolutionary change from arbuscular mycorrhization to ectomycorrhization. The ectomycorrhizal morphotypes we observed on G. opposita do not present the intraradical hyphae arrangement observed in other Nyctaginaceae. However, the absence of Hartig net recorded in our study is another indication of the plasticity and peculiarity of mycorrhizal biology of this host plant family. Of note, besides in Pisonia, the absence of Hartig net has been reported in the case of Tremelloscypha sp. and Sebacina sp. ectomycorrhizae on the roots of Achatocarpus gracilis Walter (Achatocarpaceae, Caryophyllales) in a neotropical dry forest in Mexico.61 Overall, this evidence points out the significance of Caryophyllales (that include the Nyctaginaceae) as mycorrhizal hosts in a variety of neotropical ecosystems, and the necessity to study the peculiar ectomycorrhizal associations and the role of ECM symbiosis in the Neotropics more thoroughly.62 As for G. opposita, further work is currently underway in our lab, through in vitro synthesis of ECMs with selected mycobionts, to ascertain whether the formation of Guapirioid ECMs depends on the plant host, the fungal partner, or both.\n\nâIn this ecosystem where plants need to constantly deal with various environmental stresses, the symbiotic association of plants with arbuscular mycorrhizal fungi (AMF) is one of the main strategies for their survival, due to the ability of external fungal hyphae to absorb the scarce nutrients and water from the substrate, as well as hyphae contributing to soil aggregation ⊠.and salinity tolerance,â noted da Silva and co-workers discussing the important ecological role played by AMF in restinga.63 A host of studies conducted in the restinga across Brazil since the 1990s have indeed revealed many details of the AMF communities in these ecosystems, revealing that most of coastal dune plants investigated were associated with arbuscular mycorrhiza and that restinga AMF are significantly diverse.64,65 In this context, it is relevant to note that Guapiraâand likely other restinga host plants beyondâis a dual-mycorrhizal species, capable of hosting both arbuscular mycorrhizal and ectomycorrhizal associations.66 Several ectomycorrhizal hosts share this feature, including Eucalyptus, Alnus, Populus, Salix and members of the Cistaceae.56 They are typically plants that can survive in environments that are subject to severe disruptions like natural fire or even human activity, as well as soils deficient in nutrients. The benefits of dual-mycorrhizal colonization thus stretch from plants with increased rates of survival, growth, and nutrient absorption to environments, promoting establishment and increasing survival on unfavorable locations of linked AM/ECM plants. All these considerations fit potentially well with restinga characteristics.\n\nOur attempts to assess the diversity of ectomycorrhizal fungi associated with restinga, both through direct field sampling and by surveying records in the literature and in national fungaria, revealed 726 entries (Table 1S, which can be found as Underlying data23). A total of 74 of these correspond to putative ectomycorrhizal taxa specifically associated with restinga, mostly derived from recent dedicated research and our own data (Table 1). A total of 14 different taxa were recorded in our fieldwork in restinga fragments in southern Brazil; all are reported for the first time as linked to Guapira opposita. Several important ectomycorrhizal fungal taxa are represented in the list, with Boletaceae (15 spp.), Amanita (9 spp.), Tomentella/Thelephora (8 spp.), Russula/Lactifluus (7 spp.), and Clavulina (4 spp.). Three taxa of hypogeous fungi were recorded in the restinga, including the recently described Longistriata flava Sulzbacher, Orihara, Grebenc, M.P. MartÃn & Baseia, possibly associated with Coccoloba and Guapira spp.67\n\nThroughout our investigation, we isolated 10 distinct morphotypes from restinga fragments using random soil sampling (Table 1). Basidiomata of equivalent species were also collected in two cases (Amanita viscidolutea and Austroboletus festivus). Another eight species were collected only in association with host roots (Inocybe sp., Thelephora sp. and six unknown species of Tomentella). The high frequency of Thelephoraceae representatives as fungal partners is remarkable in our data, as well as in other studies.52,68 It is widely recognized that many species in this family are saprotrophs, however, it is possible that ectomycorrhizal species also occupy niches as saprotrophs to survive periods when they are not associated with the plant symbiont.69 Previous studies indicate that many thelephoroid fungi associated with members of the Pisonieae tribe (Guapira, Neea and Pisonia, except P. grandis) are generalists, as all telephoroid fungi found associated with members of Pisonieae were also found associated with other plant symbionts.68,70 Ectomycorrhizal plants of the Polygonaceae, Caesalpiniaceae and Fabaceae families often occur in the same regions as the Pisonieae species, such that symbiosis in these species should also be examined.68â70\n\nSpecies of Entoloma, Gymnopus, Hydropus, and Phlebopus have been mentioned as ectomycorrhizal,12 possibly associated with Myrtaceae, Leguminosae, Rubiaceae, Polygonaceae, and Euphorbiaceae in the restinga.71â74 These putative ectomycorrhizal lineages, however, are not concentrated in specific clades neither form monophyletic groups of ectomycorrhizal isolates,12 in such a way that their true ectomycorrhizal status must be confirmed. The genus Boletus is not recorded from Brazil (except from exotic plantations) but several Tylopilus, Xerocomus and Phlebopus species were originally deposited under the name Boletus sp. Taking this into account, we considered for listing records of Boletus sp. only from natural habitats.\n\nDuring our survey, we unearthed notable records of ectomycorrhiza-forming fungi occurring in the restinga that deserve special mention and additional notes (Figure 3). As shown in Table 1, several boletoid taxa have been described as being associated with restinga, such as Boletellus nordestinus (MycoBank MB823951) (Figure 3a). This species has been recently described from material collected in sandy soils in the northeast of Brazil, in the states of ParaÃba and Rio Grande do Norte.75 Although only found in two locations, it is expected that this species occurs in other restinga fragments along the Brazilian Atlantic coast. However, extensive searches in southern Brazil have been conducted, and the lack of records in these areas may indicate that this is a rare species [Altielys Magnago, personal communication]. Boletellus nordestinus can be distinguished from its closely related Boletellus chrysenteroides (Snell) Snell by its dry, velutinous, chocolate brown pileus, smaller basidiospores longitudinally ridged, dichotomously forked.75 Also B. chrysenteroides is a North-American species that associates with oaks and hemlocks and grows in an unusual environment for boletus, in the midst of decayed wood.76 Although the ECM hosts of B. nordestinus are unknown, specimens have been observed growing near confirmed ectomycorrhizal host plants: Coccoloba alnifolia Casar., C. laevis Casar. (Polygonaceae) and Myrtaceae species.5 Currently, only four species of the genus are known from Brazil: Boletellus ananas var. minor Singer, B. annas var. crassotunicatus Singer have been described for the Amazon48; B. cremeovelosus Barbosa-Silva & Wartchow77 and B. nordestinus have been described for the Atlantic Forest.75\n\n(a) Boletellus nordestinus; (b) Amanita crebresulcata; (c) Amanita coacta; (d) Amanita viscidolutea; (e) Cantharellus guyanensis; (f) Clavulina junduensis; (g) Clavulina incrustata; (h) Brasilioporus olivaceoflavidus; (i) Brasilioporus simoniarum; (j) Nevesoporus nigrostipitatus. Photo credits (a) Eduardo Fazolino; (h; j) Altielys C. Magnago; (i) Juli Simon.\n\nAmanita crebresulcata (MycoBank MB308549) (Figure 3b), Amanita coacta (MycoBank MB308546) (Figure 3c) and Amanita viscidolutea (MycoBank MB514222) (Figure 3d) were also noteworthy findings from our survey. The first two are Vaginatae sect. members with a patchy distribution, with A. crebresulcata found in the states of Amazonas, Mato Grosso (in the Brazilian Amazon), ParaÃba, Paraná, Pernambuco and Santa Catarina (in the coastal Atlantic Forest),78â82 and A. coacta found in Amazonas, São Paulo and Santa Catarina [78,82,83 as A. crebresulcata]. These records, however, may not reflect the true distribution of taxa in the country, as several regions remain under-sampled, and species records are based on specialists' areas of expertise.82 Despite their macromorphological similarity, A. coacta is mainly characterized by having a felted submembranous volva, and the presence of a transverse belt-like portion of the volva that detaches from the saccade portion attached to the base of the stipe as it increases in length.84 Amanita crebresulcata, on the other hand, has a thin saccade volva that usually breaks at the apex and leaves no remnants on top of the stipe.78 Amanita viscidolutea was one of the most frequent fungi found during our field trips. Basidiomata of the species are vibrant yellow with slightly striated white pileus margin and the exannulate stipe.85 The taxon belongs to the well-supported sect. Amanita,86 reported to group important ectomycorrhizal taxa such as Amanita muscaria (L.) Lam. and Amanita pantherina (DC.) Krombh.87 The species is known from restinga areas in the coastal Atlantic Forest from Rio Grande do Norte, where it was originally described,84 ParaÃba88 and Santa Catarina.33 Although it is usually found in relatively large populations, A. viscidolutea is a rare species and it grows in a specific type of vegetation that has been threatened by habitat loss and fragmentation by human population growth and expansion, along the Brazilian coast.25 Using transmission electron microscopy, we recently described the morpho-anatomical characteristics of the ectomycorrhiza formed between A. viscidolutea and Guapira opposita roots; both partners were identified from the ectomycorrhizal root tips through molecular analyzes.33\n\nCantharellus guyanensis (MycoBank MB240517) (Figure 3e) is a widespread species that was discovered in lowland forest in French Guiana.89 Surprisingly, the species has gone nearly a century without being recorded since Montagne proposed it. However, studies of ectomycorrhizal fungi in neotropical ecosystems have revealed that the species ranges from southern Brazil to northern Colombia.90,91 Despite some minor morphological differences between the recorded specimens of C. guyanensis and the type specimen, they agree on the relevant character set. Its wide geographical distribution appears to reflect the wide variety of host plants. It can be found associated with monodominant forests of Dicymbe or Aldina spp. in Guyana; or multidiverse ectotrophic forests in spatial proximity to Coccoloba, Guapira, and Neea species in French Guiana, Colombia, Venezuela and Brazil (in the restinga).92,93 Singer et al.48 discovered C. guyanensis in the Brazilian Amazon in the 1980s, possibly associated with Aldina species as well as Glycoxylon inophyllum (Mart. ex Miq.) Ducke. Basidiomata of this species are solitary, abundant, visible only for a short period of time (for a month or so),91 have an orange yellow to orange pileus, a hymenophore clearly laminated or regularly folded at all stages of growth and the presence of purplish tints in its predominantly orange pileus.90\n\nConsidering all the Clavulina described for the restinga, Clavulina junduensis (MycoBank MB839651) (Figure 3f) deserves attention. The species is characterized by the coralloid, branched, purplish grey basidiomata with brownish orange stipe; hyaline, subglobose to broadly ellipsoid basidiospores and abundant gloepleurous hyphae with refringent content and swelling bulbs.94 Basidiomata of C. junduensis are frequently found in restinga fragments in southern Brazil but have been misidentified as Clavulina cinerea (Bull.) J. Schröt. for the past years.94â97 However, considering that specimens with dark gray coloration do not group in a single clade in the phylogenies, studies suggest C. cinerea represents a species complex and more than one species with gray coloration is subsumed under this name.98 Although we are still working on identifying the host, based on field observations, C. junduensis is possibly associated with G. opposita, which is the most common symbiont in restinga fragments in southern Brazil.33 Another species associated with the restinga that also deserves a mention is Clavulina incrustata Wartchow (MycoBank MB561193) (Figure 3g). The taxon was described by Wartchow99 based on material collected in the Atlantic Forest from Pernambuco and it is the first species of Clavulina with incrusted hyphae. The presence of crystals in the specimens represents a character of taxonomic significance within the genus, and despite being microscopically identical, Tibpromma and colleagues100 proposed Clavulina paraincrustata Meiras-Ottoni & Gibertoni to differentiate from specimens of C. incrustata with a less robust and pale basidiomata, with amphigenous hymenium. By the time of the publication, the DNA of the type specimen was tentatively extracted, but with no success.100 However, the researchers recognized that the characters used to describe the new species have low taxonomic significance and, based on new phylogenetic analyzes, they proved to be the same species and synonymization of the names is expected [Angelina Meiras-Ottoni, personal communication]. According to the available data, Clavulina is an ancestrally tropical lineage,101,102 and, although the genus can be found in a variety of ecosystems, it has been shown it is especially diverse in South America, where many new species have been recently described.11,103,104\n\nIt is known that over the years and with the expansion of molecular phylogenetic analyzes of Boletaceae, the family has undergone several re-circumscriptions, with the rescue of some taxa and the segregation of others,105,106 as in the case of the two newly proposed genera, Brasilioporus and Nevesoporus.74 Brasilioporus olivaceoflavidus (MycoBank MB836726) (Figure 3h), the type species of the genus, was collected in the coastal Atlantic Forest of the state of EspÃrito Santo, but it has also been recorded for the state of Santa Catarina, in restinga fragments.74 This species has tiny basidiomata, a pileus with fibrils and olive-green scales on a yellowish background, and a blackish hymenophore when injured. It grows solitary and in small groups, or gregarious on sandy soil in the vicinity of ectomycorrhizal Coccoloba, Guapira, and Pisonia species. Brasilioporus simoniarum (MycoBank MB836727) (Figure 3i) is a Brazilian species phylogenetically close to it. This species has been described as growing clustered in groups of three basidiomata on restinga sandy soil, in vicinity of Guapira spp. and it is known only from the type locality in the Brazilian Atlantic Forest on Florianópolis Island.74 Different from B. olivaceoflavidus, Brasilioporus simoniarum have distinguished purplish black basidiomata, fibrillose to squamulose pileus, whitish hymenophore mottled orange-red and gradually turning black and subreticulate/sublacunose stipe. A beautiful, although discreet, species associated with restinga is Nevesoporus nigrostipitatus (MycoBank MB838704) (Figure 3j), characterized by the small, basidiomata, with pinkish brown velvety pileus, pinkish tubes that are slightly depressed around the stipe and unchanging where bruised, and slender, dark gray to blackish stipe.74 Although the species has only been found in the type locality, EspÃrito Santo, and ParaÃba, it is expected to be found along the entire coast of the Atlantic Forest. In situ, Nevesoporus nigrostipitatus grows gregariously in small groups on sandy soil near species of Coccoloba and Guapira.\n\nDespite their obvious relevance for understanding nature and ecosystem change, fungi have traditionally been neglected in biodiversity conservation. However, a number of initiatives and studies have raised general attention toward the status of fungal populations across the world, increasing awareness and spurring protection actions dedicated to fungi.107,108 Restinga makes no exception, and together with the observation and description of its fungal diversity, it comes the assessment of the conservation status of several ectomycorrhizal taxa. A. viscidolutea, for example, has been reported from a handful of sites, although it is likely to be more widespread. A population decline of between 30% and 50% within the last three generations (50 years) has been suspected, based on the severe habitat decline in the area, justifying its conservation assessment as âVulnerableâ following the IUCN criteria.109 Another species determined as Vulnerable is A. festivus.110,111 Known from the coastal Atlantic Forest of Brazil (Pernambuco, Paraná and Santa Catarina states), A. festivus occurs solitary to scattered in white sandy soil under trees in restinga. âThere is concern over a decline of the habitat considering the restinga areas, as they are small highly fragmented patches open to recreational activities and tourism and there are no strict laws that restrict the use of these areas. Also, the areas in southern and north-eastern Brazil have been impacted by urban growth, threatening the last remnants of Atlantic Coast restingas. Invasion by non-native pine (Pinus elliottii) is another threat,â reads the original description of the threats menacing this species.111 Devising measures to efficiently protect threatened fungal restinga species is not a trivial matter. Generally speaking, protection of habitats where endangered macrofungi are found is pivotal for the conservation of these key microorganisms. To this aim, curbing the spread of invasive non-native species and avoiding excessive human exploitation of coastal areas are key conservation actions, especially when coupled to sound data on the distribution and population size of the macrofungi object of protection.\n\n\nConclusions\n\nRestinga mycorrhizal biology and ecology is under the spotlight, but clearly, we are just scratching the surface. Identity of host plants, host-specificity of associated mycobionts and patterns of shared mycorrhizal networks among host plants, the role played by dual mycorrhizal symbiosis, are only a few of the many aspects that demand further investigation.112 Besides enhancing our basic understanding of restinga as an ecosystem, casting light on these issues would also have practical consequences. Identifying symbionts and their effects on ecosystems, for example, will enable the development of conservation and restoration strategies for the restinga. Hopefully, this and other works will increase the awareness of researchers, providing us in the near future with fresh data coming from both fungal and botanical forays, aimed at describing the diversity of ECM fungi and associated plant ecology in restinga. Also, well-planned molecular studies examining mycorrhizal specificity at the root tip scale are bound to disclose many details of the structure and dynamics of restinga ectomycorrhizal communities.\n\n\nAuthor Contributions\n\nAll authors made substantial contributions to the conceptualization and design of the work, drafting the work and critically reviewing it for important intellectual content, as well as all authors approved the final version for publication. ANMF, MAN and ACR supervised the planning and execution of the research, including external guidance from the core team. ANMF annotated and curated the data for its initial use and later reuse, checking the overall reproducibility of the results. ANMF, ML and OC conducted phylogenetic analysis and morphological observations. MAN, ACR, ML and OC provided study materials and all other resources used as analysis tools. ACR, OC and ML secured financial support for the project that gave rise to this publication. All authors directed and coordinated the planning and execution of the research activity.",
"appendix": "Data availability\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Tomentella sp. isolate M30R173. Accession number OP819286; https://identifiers.org/ncbi/insdc:OP819286. 145\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Tomentella sp. isolate M34R198. Accession number OP819287; https://identifiers.org/ncbi/insdc:OP819287. 146\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Tomentella sp. isolate M36AR200a. Accession number OP819288; https://identifiers.org/ncbi/insdc:OP819288. 147\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Tomentella sp. isolate M36AR200b. Accession number OP819289; https://identifiers.org/ncbi/insdc:OP819289. 148\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Austroboletus festivus isolate M41CR210. Accession number OP819290; https://identifiers.org/ncbi/insdc:OP819290. 149\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Inocybe sp. isolate M51AR230. Accession number OP819291; https://identifiers.org/ncbi/insdc:OP819291. 150\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Thelephoraceae isolate M53AR235. Accession number OP819292; https://identifiers.org/ncbi/insdc:OP819292. 151\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Tomentella sp. isolate M57BR248. Accession number OP819293; https://identifiers.org/ncbi/insdc:OP819293. 152\n\nGenBank: Eukaryotic Nuclear rDNA/ITS/Restinga ectomycorrhizas/Tomentella sp. isolate M68AR291. Accession number OP819294; https://identifiers.org/ncbi/insdc:OP819294. 153\n\nFigshare: Collections of ectomycorrhizal fungi from restinga fragments on the Brazilian coast. https://doi.org/10.6084/m9.figshare.22196836. 23\n\nThis project contains the following underlying data:\n\nâ Furtado ANM et al - @F1000 Res_Supplementary Table 1.xlsx.\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgments\n\nThis article is a part of the PhD thesis of A.N.M.F. The authors thank LAMEB (UFSC, Brazil) for the support during the molecular work. We also thank the following: Dr. Mayara K. Caddah (UFSC, Brazil) for helping identify Guapira opposita; Dr. Savio T. Farias for helping during field expeditions; and those at the FLOR herbarium and fungarium (UFSC, Brazil) for the support. Thanks to Eduardo Fazolino, Altielys C. Magnago and Juli Simon for the photographs of Boletellus nordestinus, Brasilioporus olivaceoflavidus, Nevesoporus nigrostipitatus and Brasilioporus simoniarum. The first author was supported by the Coordenação de Aperfeiçoamento Pessoal de NÃvel Superior â Brazil â Finance Code 001 (CAPES-DS and PDSE fellowship grants).\n\n\nReferences\n\nMougi A, Kondoh M: Diversity of interaction types and ecological community stability. Science. 2012; 337: 349â351. Publisher Full Text\n\nvan der Heijden MGA, Bardgett RS, Straalen NM: The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol. Lett. 2008; 11: 296â310. Publisher Full Text\n\nSmith SE, Read DJ: Mycorrhizal symbiosis. New York: Academic Press; 3° ed2008; 815.\n\nBonfante P, Genre A: Mechanisms underlying beneficial plantâfungus interactions in mycorrhizal symbiosis. Nat. Commun. 2010; 1: 48. 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}
|
[
{
"id": "168665",
"date": "24 Apr 2023",
"name": "Julieta Ãlvarez Manjarrez",
"expertise": [
"Reviewer Expertise Tropical ecology and fungal systematics",
"especially ectomycorrhizal fungi"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is a very important piece of information about the fungal diversity of a natural ecosystem in Brazil. The sampling reported 726 records from ectomycorrhizal taxa with the identification of their hosts. The paper is very well written and I just added few comments to improve it:\nâIn Brazil, ectomycorrhizal fungi often have a fragmented distribution due to the lack of information about them and because they do not always have the same distribution as the host plantsâ\nIt is necessary to rephrase the two ideas of this sentences: the estimation of ECM fungi distribution is fragmented according to the current information; and how is it possible that the distribution have not the same distribution of host plants, I think you mean there is still missing information about all the ECM hosts in Brazil. Also, the citation you used to argue this sentence came from other countries but you are talking particularly from Brazil, could you add some accurate reference for Brazil ECM fungi and their hosts?\n\n\"This can result in high endemism at the species level due to the specific habitats they occupy.\"\nFor me is not clear how the lack of information, that is the main topic of the previous sentence, would produce high endemism. Please be more clear about this hypothesis.\n\n\"In 2016, Roy and coworkers reported approximately 180 species of ectomycorrhizal fungi in Brazilian native forests.15 In fact, the majority of the published studies were conducted in introduced Pinus and Eucalyptus plantations.\"\nThe writing is very confusing, first you mentioned the number of species in native forests, but then you jumped to talk about plantations. I recommend to remove the plantation sentence from this paragraph and try to write a better connection between all the ideas form this paragraph.\n\nIn Restinga definition, please provide information about the soil types.\n\nFor Figure 1 it would be more useful to be closer to the areas that have this vegetation instead of presenting all Latin American map, and indicate with a signal the places you studied.\n\nAdd the GenBank number accession of the ITS region from the plant.\n\nâThe roots were washed and carefully selected under a stereomicroscope.â - Which characteristics you observed to select the root tips?\n\nHow did you extract DNA from the ECM tips?\n\nIn Table 1, would be ideal to know the location of every fungal species collections. Also it is unclear how you add more than one host per ECM fungal species, 1) how did you obtain more than 1 host? Then, when you have a list of hosts, use the asterisk for every single host name to refers to potential host. For example: Euphorbiaceae, Fabaceae, Mimosaceae*, does that mean that just Mimosaceae is not confirmed or the whole plant list?\n\nAdd the GenBank number accession of your data in the Table 1. And add a column saying if the sequences were obtained from fruitbodies or ECM root tips.\n\nI found some plant names are taxonomically incorrect, for example Mimosaceae or Caesalpiniaceae. Confirm that all the plant species/family names you are using are correct. You can use theplantlist.org or tropicos.org. I encourage that if there is a list of plants from the restinga, provide it in a Support information or at least a link where we can read the possible host names in each plant family you mentioned.\n\nWhat is the percentage of similarity between your specimens of A. crebresulcata and GenBank? Could they be a different species?\n\nâConsidering all the Clavulina described for the restingaâ add citations\n\nAdd scale bar to each photo in Figures 2 and 3.\n\nProvide an anatomical photo from the EM of Guapira opposita and cited after âThe ectomycorrhizal morphotypes we observed on G. opposita do not present the intraradical hyphae arrangement observed in other Nyctaginaceae. However, the absence of Hartig net recorded in our study is another indication of the plasticity and peculiarity of mycorrhizal biology of this host plant family.â\n\nYou mentioned the AMF in restinga had been studied; besides G. opposita, do you know if your EM hosts also have AM in their roots?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "167497",
"date": "30 May 2023",
"name": "Roberto Flores Arzù",
"expertise": [
"Reviewer Expertise Ectomycorrhizal mushrooms of the Neotropics",
"especially in Subtropical lands. Ethnomycology. Ectomycorrhizae."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article proves the importance of the Restinga Forest of Brazil, a not well-known ecosystem, whose mycobiota astonish with the presence of ectomycorrhizal fungi, with common genera present in Northern Hemisphere and temperate regions as Tomentella and Thelephora, but also with new records of basidiomicetes in Amanita, Austroboletus, Clavulina, Inocybe and Russula. The study verifies the identity of the fungal symbionts not only by the fruit bodies but also by different ectomycorrhizhae in restinga soil and by molecular analyses using the ITS region, adequate but not exhaustive to separate fungal genera and species.\nThe results remind the mycobiota found in the Guyana Shield, a region with similar tree genera but with the description of a new mycorrhiza type: the guapiroid, formed by Guapira and some fungal genera. A good discussion is introduced about coexistence and the role of AMF and ECM fungi in the Restinga Forest, as well as an accurate review of the distribution of the South American ectomycorrhizal species found.\nThe results are very interesting for the fungal diversity found. Authors humbly say that they are âjust scratching the surfaceâ of the Brazilian Restinga Forest and it is quite possible that diversity will be larger.\n1. The work is clear, accurately presented and cites current literature with novel results for ECM fungi in South America.\n2. Appropriate study design, considering the references and the sequences used in the analyses and conclusions.\n3. Methodology sufficiently detailed and analysis provided can be replicated by others. There were no new techniques applied but a well-documented study to support the results.\n4. The conclusions are quite supported by the results due to the deep bibliographic analysis about fungi and symbiont plants in Tropical South America.\n5. Some minor changes could improve the paper as linking better the phrases in the first paragraph of Introduction.\n6. The Figure 2 could be omitted if published in references 33 or 50.\n7. Authors point correctly that genus Boletus has not been recorded in Brazil except from exotic plantations.\n8. Maybe they could say something about the two Phlebopus species cited in the database, considering that both species have an African origin?\n9. There is a good analysis for Boletellus in Brazil as well as for Amanita and Clavulina.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-317
|
https://f1000research.com/articles/12-314/v1
|
21 Mar 23
|
{
"type": "Research Article",
"title": "Virtual screening for chemical analogues similar to phytochemicals that inhibit aldose reductase in the development of diabetic microvascular complications",
"authors": [
"Filex Otieno",
"Richard Kagia",
"Richard Kagia"
],
"abstract": "Background: The polyol pathway contributes to the development of diabetic complications but can be inhibited by plant phytochemicals. This study aimed at assessing analogs of specific flavonoids that delay onset of microvascular complications with better pharmacokinetic and toxicology profiles. Methods: An in silico study design was employed. The phytochemicals luteolin and quercetin were selected. Analogs were obtained from ZINC database and prepared using Avogadro software. Docking analysis was done using AutoDock Vina embedded in Chimera. Ligand enzyme interaction was carried out using Biovia Discovery studio. Pharmacokinetic and toxicological profiling was carried out using SWISSADME and protox server. A total of 40 analogues were analyzed. Sulindac was used as the comparator besides original phytochemicals. Results: Docking analysis showed both luteolin and quercetin (-9.7) had a slightly stronger affinity for inhibiting aldose reductase compared with sulindac (-9.6). Eight analogues of luteolin and 14 analogues of quercetin showed stronger affinity with the highest registered at -10.6. Both luteolin and quercetin did not violate the Lipinski rule, had high GI absorption, did not cross the blood brain barrier nor were p-glycoprotein substrates, and inhibited CYP1A2, CYP2D6 and CYP3A4. The LD50 of luteolin (3,919 mg/kg) was high indicating excellent safety profile. Quercetin had a low LD50 (159 mg/kg). All 22 analogues exhibited similar pharmacokinetic profiles to their respective phytochemical. However, they did differ in terms of docking strength and toxicology analysis. Six out of the eight luteolin analogues had LD50=3,919 mg/kg, while the remaining had LD50=159 mg/kg. Five quercetin analogues had LD50 of 159 mg/kg, another five had LD50=3,919 mg/kg and the rest had LD50=4,000 mg/kg, while the other two had a LD50 of 5,000 mg/kg. Conclusions: In conclusion, six ZINC compounds similar to luteolin and nine similar to quercetin had stronger binding affinity for aldose reductase and superior toxicological profile compared to parent phytochemicals.",
"keywords": [
"Aldose reductase",
"Microvascular complications",
"Chimera",
"SWISSADME",
"Luteolin",
"Quercetin",
"Sulindac"
],
"content": "Introduction\n\nDiabetes mellitus (DM) is the number nine leading cause of death globally and a major reason for the increase in the number of male deaths since the year 2000 (World Health Organization, 2020). Currently, approximately half a billion people worldwide have been diagnosed with DM and cases are projected to rise above 600 million by 2045 (Saeedi et al., 2019). Furthermore, diabetes predisposes patients to developing other morbid conditions such as hypertension. Despite these facts, diabetes especially type 2 DM, is manageable and preventable. By definition, DM is a chronic metabolic disease that once diagnosed with, has a poor recovery prognosis (Sun et al., 2022). Its chronic form is associated with the development of medical emergencies (diabetic ketoacidosis (DKA), Hyperosmolar Hyperglycemic State (HHS)), progressive microvascular (retinopathy, neuropathy, nephropathy) and macrovascular (ischemic heart disease, peripheral vascular disease, stroke) complications that reduce the quality of life of patients (Chawla et al., 2016). Additionally, DM has been reported as being immunosuppressive as seen by increasing incidences of infections in patients suffering from it (Polk et al., 2021; Dryden et al., 2015, Hobizal & Wukich, 2012, Devrajani et al., 2010). Diabetes also increases the risk of developing other non-communicable conditions such as chronic kidney disease and dyslipidemias.\n\nIn diabetic complications, the pathological hallmark commonly surrounds the vasculature system leading to the development of microvascular and macrovascular complications (Eid et al., 2019). These complications are progressive in nature and with poor prognosis. Studies such as the United Kingdom Prospective Diabetes Study, reports that strict control of blood glucose levels limits microvascular disease but use of glucose-lowering agents relatively improves macrovascular outcomes (King et al., 1999). Background literatures portrays a mixed picture of development of diabetic complications; some scholars suggest microvascular and macrovascular complications occur simultaneously, while others posit that macrovascular complications occur independent of microvascular complications (Eid et al., 2019, Chawla et al., 2016). At the core of the cardiovascular system, microvessels (arterioles, capillaries, venules) form the basic functional unit. Blood moves between microvessels and macrovessels to supply cells with oxygen and nutrient and remove waste products. However, the cellular components and architecture of microvessels differ from that of macrovessels. Macrovessels primarily function as transport medium, while microvessels regulate blood pressure, control vascular permeability and optimize blood flow to the needs of the cells.\n\nIn patients with diabetes, excessive glucose due to hyperglycemia causes thickening of the capillary basement membrane and increased protein synthesis within the extracellular matrix. These changes together with advanced glycation end products (AGEs), and inflammation induces microangiopathy, which facilitates the development of microvascular complications, i.e., retinopathy, neuropathy and nephropathy (Giacco & Brownlee, 2010). Moreover, since these microvessels control blood pressure, such abnormalities lead to the development of hypertension. Thus it can be understood that macrovascular complications are independent risk factors for microvascular complications. However, there is a strong correlation between the two with other studies suggesting microvascular changes to be risk factors for macrovascular complications (Hurst et al., 2015). Common pathways leading to these complications are the formation of AGEs, induction of oxidative stress, low grade inflammation, neovascularization of vasa vasorum and the sorbitol pathway.\n\nThis study focuses on the sorbitol pathway/polyol pathway, which has been implicated in the development of microvascular complications (Yan, 2018). The development of microvascular complications follows a complex pathophysiology with inputs from several cellular biochemical pathways; one of them being the polyol pathway. This pathway does not solely and in absolution lead to the development of microvascular complications but has a major contribution and is critical for the pathogenesis of such complications as it has been observed in several studies (Yan, 2018; Mathebula, 2015; Lorenzi, 2007). The sorbitol pathway is a two-reaction step used to convert glucose to fructose as depicted in Figure 1.\n\nAldose reductase is a non-specific enzyme catalyzing the conversion of any sugar into its alcohol form. In healthy individuals, glucose affinity for aldose reductase is quite low but in hyperglycemic conditions, the increased glucose molecules in circulation increases enzyme affinity (Jannapureddy et al., 2021). The enzyme is found highly localized in specific cells such as epithelia of the lens, papilla and cortical cells in kidney, Schwann cells in peripheral nerves and islets of Langerhans in the pancreas. Ocular, neuronal, renal and pancreatic B-cells absorb glucose using insulin-independent glucose transporters (Jannapureddy et al., 2021). As such, the aforementioned conditions provide an ideal environment for microvascular complications to ensue. The intermediate product NADP+ acts as a negative feedback inhibitor of the enzyme preventing increased synthesis of sorbitol. However, the formed sorbitol is converted to fructose by sorbitol dehydrogenaseâanother non-specific enzyme leading to the final products of NADPH and fructose (Garg & Gupta, 2022). This negates the negative feedback of NADP+, thus ultimately, in equilibrium conditions, the forward reactions of the two reactions are favored. Within the specific cells, not all sorbitol is converted to fructose as the latter has not much use for cells in terms of energy production. Therefore, both fructose and sorbitol exist within the intracellular component of the cells yet they are osmotically active (Garg & Gupta, 2022).\n\nRetinopathy; within the epithelial cells of the lens, sorbitol and fructose leads to swelling of the lens due to absorption of water attributed to osmotic effects of sorbitol and fructose. The swelling causes formation of hydropic fibers that with time liquefy due to continued swelling, resulting in intrafibrillar cleft formation. These intrafibrillar clefts lead to opacities and cataracts (Moemen et al., 2020). Neuropathy; diabetic neuropathy is pathologically characterized by segmental demyelination. Schwann cells are responsible for the synthesis of myelin compounds and their incorporation around nerves. By doing so, they increase nerve impulse conduction. The pathogenic pathway of diabetic neuropathy in relation to sorbitol levels is not clearly understood. It is postulated that the excess sorbitol and fructose causes swelling of the Schwann cells, which diminishes their functional capability and in the long run causes their death. This explains the noted improvement in peripheral function if hyperglycemia is managed in acute diabetic conditions and also the chronic permanent irreversible change associated with it (Ab Hamid et al., 2021). Nephropathy; although chronic diabetes is associated with the development of nephropathy, the exact mechanism is yet to be elucidated. However, accumulation of sorbitol and fructose has been implicated in its development especially on the papilla and cortical cells (Luis-RodrÃguez, 2012).\n\nCurrently, over 600 plant species have been shown to have antidiabetic properties (Kayarohanam, 2015). Extract from these plants have phytochemical compounds that influence the glucose metabolic pathways and impart the pathways that lead to development of diabetic complications. The various phytochemicals work either independently or synergistically to exert their antidiabetic activity; thus we cannot say in absolution that they solely inhibit the polyol pathway. However, advanced technology has enabled isolation, characterization and visualization of the plant phytochemicals from the extracts. Besides, studies on the effect of such phytochemicals on the enzymes of the polyol pathway have reported specific phytochemicals with potent inhibitory effects on the enzymes. Aldose reductase has been the most targeted in studies as it is a rate-limiting step. Constituents such as quercetin, rosmarinic acid, nepetrin, mangiferin, luteolin, curcumin, ellagic acid, butein, and eugenol have potent aldose reductase inhibitory effects. Others such as ellagic acid and caffeic acid inhibit both aldose reductase and the sorbitol dehydrogenase enzymes, thus acting as sequential inhibitors. As such, this study aimed to virtually screen such phytochemicals for structurally similar compounds and assess their docking scores, pharmacokinetic profiles and toxicological profiles using computational methods as potential lead compounds.\n\n\nMethods\n\nThis study was carried out in the School of Pharmacy, Kabarak University. An in silico study design was employed for this research. Based on the literature review, several plants have been shown to inhibit the development of microvascular complications. The search terms âaldose reductaseâ, âpolyol pathwayâ, âaldose reductase inhibitionâ, âmicrovascular complicationsâ, and âflavonoidsâ were used to search for literature from the following search engines: PubChem (RRID:SCR_004284) and Google Scholar (RRID:SCR_008878). The last search was performed in August 2021. Target prediction using the online tool, swisstargetprediction, was used to assess and validate whether phytochemicals from such plants do bind to aldose reductase. Two phytochemicals, luteolin and quercetin, were selected. The predicted probability for binding to aldose reductase for luteolin and quercetin was 1.00. Sulindac was used as a comparator for this study as it is an approved aldose reductase inhibitor.\n\nStructure and canonical smiles of luteolin and quercetin were obtained from the PubChem website. The canonical smiles were used to screen online databases via the online tool SwissSimilarity for structurally similar compounds. ZINC database (RRID:SCR_006082) is an open access database with millions of chemical compounds that was selected for this study. The database is embedded within SwissSimilarity enabling the concomitant screening. Results of the screened compounds were downloaded as an excel file having canonical smiles of the various chemical compounds and their various similarity index. A total of 20 chemical analogs, for each phytochemical compound, with highest similarity index were isolated and used for further analysis. Canonical smiles of the 20 selected chemical compounds were drawn and âcleanedâ using the online tool PubChem Sketcher version 2.4 and each downloaded as a MDL molfile. Each of the downloaded compound will again be optimized using the computer program Avogadro (RRID:SCR_015983) software and minimized using UCSF Chimera v1.16 (RRID:SCR_004097).\n\nStructure of aldose reductase (PDB ID 3rx4) was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) (RRID:SCR_012820). Non-standard amino acids present in the enzyme were removed using UCSF Chimera.\n\nAutoDock Vina (RRID:SCR_011958) version 1.2.0 (Eberhardt et al., 2021; Trott & Olson, 2010) was used to dock the selected 20 analogs of each phytochemical to standard aldose reductase enzyme and their corresponding docking strength tabulated. The program is available as a computer application that can be embedded and run simultaneously with UCSF Chimera. Visualization of the complexes formed between ligand and target protein and their interaction was carried out using BIOVIA Discovery Studio v21.1.0.20298 (RRID:SCR_015651) (alternative AutoDock (RRID:SCR_012746), which is a free software that can be used)\n\nSwissADME, an online web tool, was used to analyze the pharmacokinetic profile and synthesizability of the selected 20 analogs for each phytochemical. In addition to target prediction for side effects, the other toxicology analysis carried out involved running the analogs on Protox-II (RRID:SCR_018506) server, a web tool used to analyze toxicology of compounds.\n\nSelected 20 analogs of each phytochemical compound analyzed were tabulated together with their similarity scores. Molecular docking scores, other target prediction, pharmacokinetic profiling, synthesizability and toxicological analysis results were also presented in form of tables, graphs and charts. Interpretation of the obtained data involved looking for compounds with: stronger affinities to increase efficacy, highly selective and less toxic to reduce side effects and better pharmacokinetic profile that can enhance dosing regimen and administration.\n\n\nResults\n\nTable 1 (Otieno, 2022) shows the plant species that have been shown to have antidiabetic activity and the phytochemicals that are purported to have antidiabetic activity. In addition, Table 1 shows that both luteolin and quercetin, which can be obtained from chamomile and fenugreek respectively, were predicted to have 100% probability of binding to aldose reductase.\n\nFigure 2 depicts the structures of luteolin, sulindac and quercetin. Notably both have a primary aromatic system associated with a carbonyl group (a thiocabonyl in the case of sulindac). In addition, luteolin and quercetin have a secondary aromatic ring that is substituted by polar groups. Sulindac also has a secondary aromatic ring system that is substituted with a fluorine and carboxylic group.\n\nStructures of (A) luteolin, (B) sulindac and (C) quercetin.\n\nTable 2 describes the analytic results for luteolin and its ZINC analogues. Luteolin was predicted to have a slightly stronger binding affinity (-9.7) for aldose reductase enzyme than sulindac (-9.6). Eight out of the 20 analogues of luteolin had docking below both sulindac and luteolin with ZINC000004349582 having the strongest predicted binding strength (-10.5). Moreover, all ZINC analogues were ⥠99.8% similar to luteolin. Luteolin did not violate the Lipinski rule of five and had a high GI absorption. Additionally, luteolin was predicted to neither cross the blood brain barrier nor be a substrate of p-glycoprotein (p-gp) (also known as MDR1, ABCB1 or CD243). Although luteolin was predicted to not inhibit CYP2C19 and CYP2C9, they inhibited CYP1A2, CYP2D6 and CYP3A4. On the contrary, the comparator inhibited all cytochrome enzymes analyzed except CYP1A2 and CYP2D6. All eight analogues of luteolin had similar pharmacokinetic profiles to their parent phytochemical compounds due to high structural similarity. On toxicological analysis, luteolin was predicted to be safer, falling under toxicology class 5 (LD50=3,919 mg/kg). Two out of the eight luteolin analogues also fell under toxicology class 3, while the rest were under class 5 with similar predicted LD50 values\n\nGI, gastrointestinal; p-gp, p-glycoprotein; BBB, blood brain barrier.\n\nTable 3 describes the analytic results for quercetin and its ZINC analogues. Quercetin was predicted to have a slightly stronger binding affinity (-9.7) for aldose reductase enzyme than sulindac (-9.6). Analysis of quercetin analogues showed that 14 out of the 20 selected ZINC compounds had better binding affinity than both parent phytochemical and the comparator. In particular, the compounds ZINC000000039111, ZINC000575623588, and ZINC000000057845 had the highest docking scores (-10.6). Furthermore, all 14 analogues were ⥠99.7% similar to quercetin. Pharmacokinetic profile prediction quercetin and the comparator (sulindac) did not violate the Lipinski rule of five and had a high GI absorption. Quercetin was predicted to neither cross the blood brain barrier nor be a substrate of p-gp (also known as MDR1, ABCB1 or CD243). Quercetin did not inhibit CYP2C19 and CYP2C9 but was an inhibitor of CYP1A2, CYP2D6 and CYP3A4. To the contrary, the comparator inhibited all cytochrome enzymes analyzed except CYP1A2 and CYP2D6. All 14 analogues of quercetin had similar pharmacokinetic profiles as their parent phytochemical compounds due to high structural similarity. On toxicological analysis, quercetin was relatively unsafe, falling under toxicology class 3 (LD50=159 mg/kg). Five quercetin analogues had LD50 of 159 mg/kg, while another five had LD50=3,919 mg/kg. Two of the remaining four quercetin analogues had LD50=4,000 mg/kg, while the other two had a LD50 of 5,000 mg/kg. Quercetin analogues with highest predicted binding affinity: compound ZINC000000039111 and ZINC000575623588 had an LD50 of 159 mg/kg, while ZINC000000057845 had an LD50 of 4,000 mg/kg.\n\nGI, gastrointestinal; p-gp, p-glycoprotein; BBB, blood brain barrier.\n\nFigure 3 displays the pictorial representation of aldose reductase in a 3D model. Figure 3A shows the peptide chains (as different colors) that constitute the entire enzyme protein. Figure 3B displays the hydrophobic surface of the active binding site of aldose reductase. Figure 3C shows how the molecule sulindac fits into the binding pocket, while Figure 3D shows the bond interaction between sulindac and enzyme amino acids.\n\n(A) 3D peptide chains of the enzyme aldose reductase. (B) Hydrophobic surface of active binding pocket of aldose reductase. (C) Sulindac fitting in the hydrophobic binding pocket of aldose reductase. (D) Bond interaction between sulindac atoms and amino acids present on the walls of active binding pocket of aldose reductase.\n\nFigure 4 shows the interaction of aldose reductase enzyme with luteolin and its highest binding analogue, ZINC000004349582. Figures 4A and 4C show how luteolin and ZINC000004349582 fits into the binding pocket, while Figures 4B and 4D show the bond interaction between enzyme proteins and luteolin (Figure 4B) and ZINC000004349582 (Figure 4D).\n\n(A) Luteolin fitting in the hydrophobic binding pocket of aldose reductase. (B) Bond interaction between luteolin atoms and amino acids present on the walls of active binding pocket of aldose reductase. (C) ZINC000004349582 fitting in the hydrophobic binding pocket of aldose reductase. (D) Bond interaction between atoms in compound ZINC000004349582 and amino acids present on the walls of active binding pocket of aldose reductase.\n\nFigure 5 shows the interaction of quercetin and its highest binding analogue, ZINC000000057845, with aldose reductase enzyme. Figures 5A and 5C show how quercetin and ZINC000000057845 fits into the binding pocket, while Figures 5B and 5D show bond interactions between enzyme proteins and quercetin (Figure 5B) and ZINC000000057845 (Figure 5D).\n\n(A) Quercetin fitting in the hydrophobic binding pocket of aldose reductase. (B) Bond interaction between quercetin atoms and amino acids present on the walls of active binding pocket of aldose reductase. (C) ZINC000000057845 fitting in the hydrophobic binding pocket of aldose reductase. (D) Bond interaction between atoms in compound ZINC000000057845 and amino acids present on the walls of active binding pocket of aldose reductase.\n\n\nDiscussion\n\nSeveral plants have been shown to specifically inhibit aldose reductase enzyme, which is implicated in the development of diabetic complications (Saraswat et al., 2008). Further, synthetic derivatives have also been formulated but remain unsuccessful when it comes to clinical trials (Singh Grewal et al., 2015). As such, none are yet approved as medication for the management of either retinopathy, nephropathy or neuropathy. Aldose reductase inhibitors (ARIs) have been shown to bind at a separate site from that of NADPH and glucose, which is highly hydrophobic as presented in Figure 3B. Studies on structural activity relationships show that ARIs need to have a primary lipophilic moiety, often an aromatic ring and a thiocarbonyl or carbonyl group that is located 2.8 to 3.8A from the center of the primary group. Acetylsalicylic acid and sulindac (Figure 2B) conform to such characteristics but higher concentrations than the therapeutic range are required to inhibit the enzyme. The addition of a second lipophilic group has been shown to increase inhibitory activity as seen in compounds such as quercetin (Figure 2C) even at micro molar concentrations (in general flavonoids) (Kawanishi et al., 2003). The phytochemicals analyzed in this study were thus from the flavonoid group, luteolin and quercetin. Both phytochemicals were predicted to have 100% probability of binding the enzyme as presented in Table 1.\n\nComparatively, both luteolin and quercetin compounds were predicted to have a slightly stronger binding affinity (-9.7) for aldose reductase enzyme than sulindac (-9.6) as presented in Tables 2 and 3. A docking score of below -8.0 is generally taken as better binding strength as depicted by both compounds. Eight out of the 20 analogues of luteolin had docking below both sulindac and the parent phytochemical, with ZINC000004349582 having the strongest predicted binding strength (-10.5) as shown in Table 2. Moreover, all compounds were 99.8% or above similar to luteolin. Analysis of quercetin analogues showed that 14 out of the 20 selected ZINC compounds had better binding affinity than both parent phytochemical and the comparator. In particular, the compounds ZINC000000039111, ZINC000575623588, and ZINC000000057845 had the highest docking scores (-10.6) even slightly higher than those of luteolin. Furthermore, their similarity index was above 99.7% as seen in Table 3.\n\nLuteolin, quercetin, sulindac, ZINC000004349582 and ZINC000000057845 all fit within the hydrophobic binding pocket of aldose reductase as shown in Figures 4A, 5A, 3C, 4C and 5C, respectively. The hydrophobic pocket structural analysis of the complexes formed between the ligands and the enzyme showed that the benzaldehyde-carbonyl moiety in sulindac (Figures 3C and 3D) interacts with the amino acids valine-148 and isoleucine-156 of the peptide chain. Conversely, the chromene ring in both luteolin (Figure 2) and quercetin (Figure 4) has the keto substituent bearing the carbonyl that interacts with methionine-144 (Figures 4B and 5B). The valine-148 that interacted with carbonyl moiety in sulindac (Figure 3D) interacts with the benzyl portion of chromene ring in both phytochemicals (Figures 4B and 5B). The substituted phenyl ring serves as the second lipophilic group that is proposed to increase inhibitory activity seen in flavonoids. However, structural analysis of luteolin and quercetin best analogues, showed that the chromene keto group interacts with leucine-104 (Figure 4D) and aspartate-102 (Figure 5D), respectively.\n\nMore interactions of the analogues with aldose reductase are seen with the substituted phenyl ring attached to the chromene ring. In particular, the hydroxyl groups and the hydrogen atoms in both analogues may interact with any of the following amino acids: leucine-104, leucine-152, valine-153, tyrosine-103, alanine-155, and lysine-154. In summary, peptide amino acids running between the 100 and 150 position form active site of such molecules and may interact with them.\n\nPharmacokinetic profile prediction showed that luteolin (Table 2), quercetin (Table 3) and the comparator (sulindac) did not violate the Lipinski rule of five and had a high GI absorption probably due to the ring systems that cancel out the hydrophilic nature of the attached group, thus making the molecules relatively neutral. Additionally, both phytochemicals were predicted to neither cross the blood brain barrier nor be a substrate of p-gp (also known as MDR1, ABCB1 or CD243) as presented in Tables 2 and 3, respectively. The p-gp protein functions to efflux drugs into the intestinal lumen, reducing absorption and bioavailability (Amin, 2013). Though both phytochemicals were predicted to not inhibit CYP2C19 and CYP2C9, they were however inhibitors of CYP1A2, CYP2D6 and CYP3A4. On the contrary, the comparator inhibited all cytochrome enzymes analyzed except CYP1A2 and CYP2D6. All the eight and 14 analogues of luteolin and quercetin had similar pharmacokinetic profiles as their parent phytochemical compounds due to high structural similarity. As such, the ZINC molecules could induce potential drug-drug interaction via the prevention of other drug metabolism.\n\nOn toxicological analysis, luteolin was predicted to be safer, falling under toxicology class 5 (LD50=3,919 mg/kg), while both quercetin and sulindac were relatively unsafe, falling under toxicology class 3 (LD50=159 mg/kg and LD=264 mg/kg, respectively) (Gadaleta et al., 2019). Two out of the eight luteolin analogues also fell under toxicology class 3, while the rest were under class 5 with similar predicted LD50 values, as shown in Table 2. In comparison, five quercetin analogues had LD50 of 159 mg/kg, while another five had LD50=3,919 mg/kg. Two of the remaining four quercetin analogues had LD50=4,000 mg/kg, while the other two had a LD50 of 5,000 mg/kg, as presented in Table 3. The luteolin analog with the strongest binding affinity had an LD50 value of 3,919 mg/kg, while for quercetin the best docking compounds, compound ZINC000000039111 and ZINC000575623588, had an LD50 of 159 mg/kg, while ZINC000000057845 had an LD50 of 4,000 mg/kg.\n\n\nConclusions\n\nIn conclusion, luteolin and quercetin had better docking scores and, thus, higher binding strength compared with sulindac. A total of eight out of the 20 luteolin analogues had docking scores more negative than parent phytochemical compound, and 14 out of the 20 quercetin analogues had docking scores more negative than parent phytochemical compounds. Luteolin analogue (ZINC000004349582) and quercetin analogues (ZINC000000039111, ZINC000575623588, and ZINC000000057845) had the most negative scores (-10.5 and -10.6, respectively) and thus the strongest predicted binding affinity. Both phytochemicals and the eight and 14 analogues had similar pharmacokinetic profiles, with all obeying the Lipinski rule, having a high GI absorption, neither crossing the blood brain barrier nor being acted upon by p-gp and were inhibitors of CYP1A2, CYP2D6 and CYP3A4. Luteolin was predicted to be relatively safer than both quercetin and sulindac. Most analogues of luteolin were generally safer, while the majority of quercetin analogues had greater LD50 values compared with luteolin.\n\nBased on the aforementioned discussion and conclusions, we recommend an in vitro study be carried out to assess and validate results obtained from this in silico study.",
"appendix": "Data availability\n\nHarvard Dataverse: VIRTUAL SCREENING FOR CHEMICAL ANALOGUES SIMILAR TO PHYTOCHEMICALS THAT INHIBIT ALDOSE REDUCTASE IN THE DEVELOPMENT OF DIABETIC MICROVASCULAR COMPLICATIONS. https://doi.org/10.7910/DVN/3Y2SSD (Otieno, 2022).\n\nThis project contains the following underlying data:\n\n- New folder.rar (Contains data on: how the phytocompounds, their analogues and sulindac were docked to the enzyme aldose reductase and the complexes formed. The file types contained in the zipped file are CONF, .PDB, .PDBQT, .MOL2, AND.SDF. All file types can be opened with Chimera software)\n\n- Project analysis.xlsx (Docking score results, pharmacokinetic and toxicological analysis of zinc analogues)\n\n- sulindac then luteolin then quercetin ADME.tab (Pharmacokinetic analysis from SwissADME for parent phytocompounds and comparator)\n\n- swissadme of analogues (1).xlsx (Pharmacokinetic analysis from SwissADME for zinc analogues of parent phytocompounds)\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\nWe presented this research at the 2nd Global Webinar on Diabetes and Endocrinology conference held on November 28-29, 2022.\n\n\nReferences\n\nAb Hamid N, Omar N, Ismail CA, et al.: Insight of mechanism and signaling pathway in pathogenesis of diabetic neuropathy: A review. Int. Med. J. Malays. 2021; 20(4). Publisher Full Text\n\nAmin ML: P-glycoprotein inhibition for optimal drug delivery. Drug Target Insights. 2013; 7: DTI.S12519âDTI.S12534. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChawla R, Chawla A, Jaggi S: Microvasular and macrovascular complications in diabetes mellitus: Distinct or continuum? Indian J. Endocrinol. Metab. 2016; 20(4): 546â551. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDevrajani B, Shah SZ, Soomro A, et al.: Type 2 diabetes mellitus: A risk factor for helicobacter pylori infection: A hospital based case- control study. Int. J. Diabetes Dev. Ctries. 2010; 30(1): 22â26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDryden M, Baguneid M, Eckmann C, et al.: Pathophysiology and burden of infection in patients with diabetes mellitus and peripheral vascular disease: Focus on skin and soft-tissue infections. Clin. Microbiol. Infect. 2015; 21: S27âS32. PubMed Abstract | Publisher Full Text\n\nEberhardt J, Santos-Martins D, Tillack A, et al.: AutoDock vina 1.2.0: New docking methods, expanded force Field, and Python bindings.2021. Publisher Full Text\n\nEid S, Sas KM, Abcouwer SF, et al.: New insights into the mechanisms of diabetic complications: Role of lipids and lipid metabolism. Diabetologia. 2019; 62(9): 1539â1549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGadaleta D, VukoviÄ K, Toma C, et al.: SAR and QSAR modeling of a large collection of LD50 rat acute oral toxicity data. J. Cheminformatics. 2019; 11(1): 58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarg SS, Gupta J: Polyol pathway and redox balance in diabetes. Pharmacol. Res. 2022; 182: 106326. PubMed Abstract | Publisher Full Text\n\nGiacco F, Brownlee M: Oxidative stress and diabetic complications. Circ. Res. 2010; 107(9): 1058â1070. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHobizal KB, Wukich DK: Diabetic foot infections: Current concept review. Diabetic Foot & Ankle. 2012; 3(1): 18409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHurst C, Thinkhamrop B, Tran HT: The association between hypertension comorbidity and Microvascular complications in type 2 diabetes patients: A nationwide cross-sectional study in Thailand. Diabetes Metab. J. 2015; 39(5): 395â404. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJannapureddy S, Sharma M, Yepuri G, et al.: Aldose reductase: An emerging target for development of interventions for diabetic cardiovascular complications. Front. Endocrinol. 2021; 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKawanishi K, Ueda H, Moriyasu M: Aldose reductase inhibitors from the nature. Curr. Med. Chem. 2003; 10(15): 1353â1374. Publisher Full Text\n\nKayarohanam S: Current trends of plants having Antidiabetic activity: A review. J. Bioanal. Biomed. 2015; 07(02). Publisher Full Text\n\nKing P, Peacock I, Donnelly R: The UK prospective diabetes study (UKPDS): Clinical and therapeutic implications for type 2 diabetes. Br. J. Clin. Pharmacol. 1999; 48(5): 643â648. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLorenzi M: The Polyol pathway as a mechanism for diabetic retinopathy: Attractive, elusive, and resilient. Exp. Diabetes Res. 2007; 2007: 1â10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuis-RodrÃguez D: Pathophysiological role and therapeutic implications of inflammation in diabetic nephropathy. World J. Diabetes. 2012; 3(1): 7â18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMathebula SD: Polyol pathway: A possible mechanism of diabetes complications in the eye. Afr. Vis. Eye Health. 2015; 74(1). Publisher Full Text\n\nMoemen LA, Abdel Hamid MA, Wahab SA, et al.: Role of advanced glycation end products and sorbitol dehydrogenase in the pathogenesis of diabetic retinopathy. Bulletin of the National Research Centre. 2020; 44(1). Publisher Full Text\n\nOtieno F: Virtual Screening for Chemical Analogues Similar to Phytochemicals That Inhibit Aldose Reductase in The Development of Diabetic Microvascular Complications. [Dataset]. Harvard Dataverse. 2022; V1. Publisher Full Text\n\nPolk C, Sampson MM, Roshdy D, et al.: Skin and soft tissue infections in patients with diabetes mellitus. Infect. Dis. Clin. N. Am. 2021; 35(1): 183â197. Publisher Full Text\n\nSaeedi P, Petersohn I, Salpea P, et al.: Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the international diabetes Federation diabetes atlas, 9th edition. Diabetes Res. Clin. Pract. 2019; 157: 107843. PubMed Abstract | Publisher Full Text\n\nSaraswat M, Muthenna P, Suryanarayana P, et al.: Dietary sources of aldose reductase inhibitors: prospects for alleviating diabetic complications. Asia Pac. J. Clin. Nutr. 2008; 17(4): 558â565. PubMed Abstract\n\nSingh Grewal A, Bhardwaj S, Pandita D, et al.: Updates on aldose reductase inhibitors for management of diabetic complications and non-diabetic diseases. Mini-Rev. Med. Chem. 2015; 16(2): 120â162. PubMed Abstract | Publisher Full Text\n\nSun H, Saeedi P, Karuranga S, et al.: IDF diabetes atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. Diabetes Res. Clin. Pract. 2022; 183: 109119. PubMed Abstract | Publisher Full Text\n\nTrott O, Olson AJ: AutoDock vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010; 31: 455â461. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: WHO reveals leading causes of death and disability worldwide: 2000-2019. WHO|World Health Organization;2020, December 9.Reference Source\n\nYan L: Redox imbalance stress in diabetes mellitus: Role of the polyol pathway. Animal Models and Experimental Medicine. 2018; 1(1): 7â13. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "204447",
"date": "08 Feb 2024",
"name": "Ajmer Singh Grewal",
"expertise": [
"Reviewer Expertise Drug design"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is a valuable contribution to diabetic microvascular complications research, utilizing in silico methods to screen chemical analogues of luteolin and quercetin, two known aldose reductase inhibitors. The identification of analogues with stronger binding affinity and improved toxicological profiles than the parent phytochemicals is promising. Comprehensive pharmacokinetic assessments and safety evaluations, including high LD50 values for some analogues, offer a sound basis for further experimental validation and potential drug development. These findings advance the field by providing candidates with enhanced therapeutic potential, offering hope for more effective treatments for diabetic microvascular complications. To enhance the impact and applicability of this research, it is crucial to conduct rigorous experimental validation, including in vitro and in vivo studies, to corroborate the in silico findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "228134",
"date": "08 Feb 2024",
"name": "Jainey James",
"expertise": [
"Reviewer Expertise In silico study",
"synthetic chemistry"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe quantum of work can be increased further.\n\nMore recent citations can be added from in silico field.\n\nThe reason for selecting the phytocompounds can be detailed. The binding energy calculations should be incorporated, and appropriate methods should validate docking scores.\n\nMaterials and methods have to be reframed under subheadings.\n\nThe scope for future work should be emphasized.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-314
|
https://f1000research.com/articles/11-1431/v1
|
05 Dec 22
|
{
"type": "Research Article",
"title": "A cross-sectional survey among parents to report challenges and barriers in the administration of drugs to children",
"authors": [
"Nour Aliyan AlKaddour",
"Rawa Banoori Shah",
"Syed Wasif Gillani",
"Niloofar Hadi Sharafi",
"Aiman Fatima Khan",
"Riham Mohamed Elshafie",
"Hassaan Anwer Rathore",
"Nour Aliyan AlKaddour",
"Rawa Banoori Shah",
"Niloofar Hadi Sharafi",
"Aiman Fatima Khan",
"Riham Mohamed Elshafie",
"Hassaan Anwer Rathore"
],
"abstract": "Background: Lack of knowledge among parents can result in inappropriate administration practices. After analyzing different studies among children, there was no data on challenges and barriers in the administration of drugs among children in this region because of the diverse environmental issues and challenges in the UAE. The objective of this study was to determine the reported administration practices of parents and challenges and barriers in the administration of drugs among children in UAE. Methods: A questionnaire-based survey was conducted. A convenience sampling technique was used to collect the data. An online Raosoft® sample size calculator was applied (n = 248). The inclusion criteria were parents who had a child under 10 years of age and gave consent to participate in this study. Children with vision problems, cognitive/physical disabilities, and caregivers other than parents were excluded from this study. Results: The study reported response rate of 73.2%. The mean ± S.D age of the parents in years was 35.5 ± 7.8, and the mean ± S.D of children aged years was 2.60 ± 1.54. The majority of parents (83.9%) completing the survey were mothers and resided in the city (97.2%). When the children did not like taking tablet drugs 41.9% used multiple practices and 26.2% of parents reported treatment failure due to oral drug administration. Around 47.6% of those who were interviewed reported that their children had swallowing problems during the administration of oral medication. A total of 22.2% of parents reported that they gave drugs in doses higher than prescribed by the doctor to treat their children more quickly. Similarly, a total of 64.5% of the parents reported self-medication without consultation from a healthcare provider. Conclusions: The study concluded that there were inappropriate drug administration practices among parents. Parents reported administration of higher doses to treat their children quickly.",
"keywords": [
"children",
"parentsâ perceptions",
"drug administration",
"pediatric dosing",
"swallowing problems"
],
"content": "Introduction\n\nThe literature shows that infectious diseases are the foremost common reason behind significant morbidity and mortality in early childhood; this is similar in developed economies where populations have high-quality housing and access to high-quality medical care.1 In Africa, the top three fatalities of children under the age of five are pneumonia, diarrhea, and malaria. For children from low-income families, primary prevention of these illnesses is difficult. To reduce child deaths, accurate identification and rapid treatment with good therapy are critical.2\n\nUneducated mothers and children delivered at home had lower healthcare utilization for diarrhea and cough.3 Some long-standing issues have persisted, resulting in unacceptably high illness rates. Because of a lack of worldwide funding, feasible control methods have only lately been established. Vaccine-preventable diseases continue to be poorly controlled in many regions of the developing world.4\n\nOver-the-counter medications have become a significant issue in children. The absence of parental training, poor counseling, and lack of knowledge are the leading cause of drug errors. Some studies also showed that the parents did not use the correct equipment to administer the medications.5 According to the findings, a considerable majority of parents did not use the proper equipment to deliver drugs, utilized non-prescription pharmaceuticals, did not administer medications at the appropriate intervals, and blended medication into foods.6\n\nOne of the practices in pediatrics is to mix the medicine with food. The use of bodily strength is known as forced administration. When children have a low tolerance for unpleasant tastes, open administration is used. Many parents crushed the capsules or tablets, mixed them with yogurt or meal, or dissolved them in water.7\n\nAfter analyzing different studies among children, there was no data on challenges and barriers in the administration of drugs among children in this region because of the diverse environmental issues and challenges in the UAE. The objective of this study is to determine the parents reported administration practices and challenges and barriers in the administration of drugs among children in the UAE.\n\n\nMethods\n\nThis study used an observation survey design to evaluate the objective. This study will be conducted in public places, in addition to the outpatient department of a tertiary care hospital, in Ajman, UAE. The data was collected over seven months from October 2021 to April 2022.\n\nA questionnaire-based survey was conducted to assess parents' reported challenges and barriers in administration of medicine to their children at home. It was pre-validated and adopted from the literature.\n\nPart one of the questionnaire consisted of demographic information together with sex, residency, age, occupational status, married status, academic level, monthly financial gain, and a variety of children between six months and ten years.\n\nPart two collected information about medications, including the following questions: who is responsible for administering medications at home, whether the child has ever refused to take tablets/pills, what to do if the child does not like taking tablets, and whether the treatment process fails because of their child do not like to take tablets, whether the treatment process failed because the child did not like to take liquid medicine and the source of information provided to the child on the medicine.\n\nPart three consisted of information about the child, including whether the child has difficulty swallowing drugs, the types of swallowing problems, the number of times they complain about dysphagia, whether they have discussed dysphagia with the doctor, and the doctorâs recommendations.\n\nPart four consisted of information about the practice, including questions about the tools you use to give your child prescription drugs, whether you have read the leaflet attached to the drug, whether the dose given to the child is higher than the prescription drug used to treat the child quickly, whether to give the child more than one, oral medicine at the same time, whether to give the child medicine without a doctor's prescription, the type of medicine used, whether the time when the child was given the medicine is recorded, when the child recovers, do the remaining drugs What's the deal. If the medicine is prescribed three times a day, they are also required to provide time for the child's medicine. All the parts of the questionnaire consist of âyes/noâ and multiple-response questions.8\n\nFor this study's sample size, a convenience sampling technique was used to collect the data. An online Raosoft sample size calculator is applied to determine the sample size, which was 339. By assuming that the margin of error is 5%, CI 95%, a population size of 20000, and response rate of parents is 66%, according to the study conducted in UAE.9\n\nThe inclusion criteria were parents who had a child under ten years of age and gave consent to participate in this study. And also, parents who had children with or without acute illnesses such as bronchitis, malaria, pneumonia, diarrhea, respiratory disorders, and cough, were responsible for administering medication to their children. Children with vision problems, cognitive/physical disabilities, and caregivers other than parents were excluded from this study.\n\nThe study was approved by the Gulf Medical University Institutional Research Board (IRB) (Reference number: IRB/COP/STD/86/Oct-2021). The questionnaire content was described before giving it to the parents.\n\nA written consent form was obtained from all the participants before they participated in the study.\n\n\n\nâ Parents' practices during the administration of oral & liquid drugs to their children at home and the acceptable behaviors of their children, how the parents overcome administration obstacles.\n\nâ Reported problems during the administration.\n\nâ Sources of parents' information.\n\nâ Prevalence for usage and type of non-prescribed drugs.\n\nThe data was coded, categorized, and entered into the Statistical Package for the Social Sciences (SPSS). The sociodemographic and clinical data are represented using descriptive statistics (frequency, percentage, mean, standard deviation).\n\n\nResults\n\nThe study reported a response rate of 73.2%. A total of 399 participants were invited, among them, 248 consented and completed the survey questionnaire.\n\nTable 1 shows the demographics of our research participants' parents. The mean ± SD age of parents at one was 35.5 ± 7.8, and the mean ± SD of children aged years was 2.60 ± 1.54. The majority of parents (83.9%) who completed the survey were mothers and resided in the city (97.2%). Among them, 62.9% had university-level education.\n\nMothers accounted for 91.9% of those in charge of drug administration at home (Table 2). Over half of parents surveyed said they didn't try to give their children tablets when asked about their children's acceptance behavior during oral drug administration. When their children did not like taking tablet drugs, 41% utilized several techniques, and 17% convinced their children to drink more water. Twenty-two percent of parents reported treatment failure due to oral drug administration, and 14% requested another form (Table 2).\n\n* Health care provider.\n\na Percentage was calculated by dividing by 117 âthe number of children refused taking capsulesâ.\n\nb Percentage was calculated by dividing by 109 âthe number of the children refused to take liquid drugsâ.\n\nForty-four percent of parents reported that children refused liquid medications, fifty-point-five percent used different practices, and eleven-point nine percent forced their children to take the liquid drug. Twenty-four points two failed to administer the treatment.\n\nAs indicated in Table 2, the majority of parents (58.5%) got information on the drug from numerous sources, in addition to doctors (27.3%), the Internet (2.4%), and pharmacists (1.4%).\n\nAround 47.6% of those who were interviewed reported that their children had swallowing problems during the administration of oral medication, where multiple difficulties were the most common in 40.7% of the cases. Of those who reported swallowing problems, 85.6% percent discussed the problem with their doctor, who advised them to change the drug in most cases (32.7%) or advice to overcome the problem (30.6%) (Table 3).\n\na Percentage calculated by dividing by 101 âthe number of parents discussed swallowing problem with their doctorâ.\n\nA total of 32.7% of parents used multiple tools and cups attached to drugs to administer oral liquid drugs; however, other tools were also used (Table 4). A section of parents (22.2%) reported that they gave drugs in doses higher than prescribed by the doctor to treat their children more quickly. Almost two-thirds of the parents (46%) said that they disposed of the residual amount of the drug when the child recovered, while 53.6% kept it for later use.\n\nSurprisingly, sixty-four-point five percent of parents used drugs without a prescription from a doctor. Multiple drugs (104, 41.9%) are the most commonly used self-therapies, antipyretics (n:45,18.1%) (Table 5). In the final part of the survey, the parents were asked about the interval that should be left between each dose, when a drug is prescribed to be given three times daily, and it was revealed that 9.7% administered medication incorrectly.\n\n* Antipyretics/Antibiotics/Antidiarrheal/Laxatives/Antiemetic/Cough drugs/Colic drugs/Creams/Influenza drugs.\n\n\nDiscussion\n\nThis study looks at parents' practices when giving their children oral medication at home. Our research uncovered incorrect practices such as self-medication practices, using multiple drugs, higher dose administration, and inappropriate administration tools, in addition to obstacles, for example, multiple swallowing difficulties and treatment failures.\n\nMany factors, including the disagreeable taste, can impact a child's acceptance and adherence to their prescriptions, and this can cause problems for parents when providing medications to their children.10 In our study, about 47.2% of parents said their children disliked taking oral medicines, in addition to 26.2 % of tablet treatment failure. Parents try a variety of solutions to solve the problem, including mixing the medicine with milk or their children's favorite food or drink. 6 percent of parents in our survey tried mixing tablets with food, 7.7 percent dissolved tablets in water or other drinks, liquid treatment failure was 24.2, 10.1 percent tried mixing liquid drugs with juice, and 2.8 percent mixed with food and milk. When medications are used with particular foods, drug interactions and drug absorption may be affected.11,12\n\nRabia Bushra and Nousheen Aslam, conducted a review on food-drug Interactions. After single and frequent doses of Coca-Cola, the Cmax and AUC0-alpha of ibuprofen were dramatically enhanced, indicating improved ibuprofen absorption. When taking ibuprofen with Coca-Cola, the daily dosage and frequency must be lowered.13\n\nOne should note here that there might be a correlation between parents' practice in our study regarding 41.9% multiple drugs self-medication by parents for their children and high prevalence of self-medication with antibiotics (53%) and sedative/hypnotics (27%) was also observed among high school students in UAE.14,15 Self-medication, especially non-responsible self-medication, is far from being a perfectly safe activity. Incorrect self-diagnosis, delays in obtaining medical counsel when needed, and occasional but severe adverse reactions are all potential dangers of self-medication.16 Reye syndrome is a potentially fatal aspirin reaction in young infants. To avoid major adverse medication reactions, many parents are unaware that aspirin should not be administered to children under the age of 12 and should be used very carefully or not at all in adolescents aged 12â16.17\n\nFive-point one percent crush, open and break the capsules to administer them to their children. Soft gelatin capsules containing liquid should not be chewed or split since the liquid inside could be extracted, resulting in an improper dosage. Crushing drugs may lead to side effects and toxicity.18\n\nDose errors are prevalent because dosing for children must be determined individually depending on the patient's age and weight. Non-standardized teaspoons and tablespoons lead to measurement errors.19 In our study, multiple tools were used (32.7%), while 10.1% of parents used teaspoons and 3.2% used tablespoons. To reduce drug errors, the findings suggest a milliliter-only norm.20 Acetaminophen is the most commonly prescribed pediatric analgesic and antipyretic. There are numerous accounts of significant morbidity and mortality with repeated supratherapeutic doses in the literature.21 Furthermore, in our article, 22.2% of the parents gave their children drugs in higher quantities than the doctor suggested in an attempt to treat their children faster.\n\nThere are various drawbacks to this study. For starters, having the researcher present while answering questions may introduce uncontrollable biases. Second, because this is a cross-sectional study, causal links between variables were not possible to establish. Third, the use of convenience sampling may have skewed the results. Finally, in terms of swallowing problems, the main limitation of the existing study is the absence of data about the child's age, which is critical for distinguishing between drug-sophisticated children and drug-naive children.\n\n\nConclusions\n\nThe study concluded that the parents practice inappropriate drug administration. It is also found that parents frequently administered higher doses to treat their children quickly. Self-medication using multiple drugs is a substantial concern.\n\nMedication errors must be made more visible to parents. It is suggested that an intervention be designed for educational programs to educate parents about drug administration practices. When a doctor writes a prescription that must be taken multiple times per day, the intervals between doses should be indicated in hours. The primary sources of drug information should be the doctor and pharmacist. Finally, more stringent laws make it illegal to use antibiotics without a prescription.\n\n\nConsent\n\nWritten informed consent for publication of the participantsâ details was obtained from the participants.",
"appendix": "Data availability\n\nFigshare: Underlying data for âA cross-sectional survey among parents to report challenges and barriers in the administration of drugs to childrenâ, https://doi.org/10.6084/m9.figshare.20208938.v1. 8\n\n⢠Data file: Parents reported challenges and barriers in the administration of drugs among children.xlsx (Questionnaire responses)\n\nFigshare: Extended data for âA cross-sectional survey among parents to report challenges and barriers in the administration of drugs to childrenâ, https://doi.org/10.6084/m9.figshare.20208938.v1. 8\n\n⢠Supplementary file: Parents reported challenges and barriers in the administration of drugs among children.pdf (Questionnaire)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nReferences\n\nLambert SB, Ware RS, Cook AL, et al.: Observational research in childhood infectious diseases (orchid): A Dynamic Birth Cohort Study. BMJ Open. British Medical Journal Publishing Group;2012; 2(6): e002134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKahabuka C, KvÃ¥le G, Hinderaker SG: Care-seeking and management of common childhood illnesses in Tanzaniaâresults from the 2010 Demographic and Health Survey. PLoS One. 2013; 8(3): e58789. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlene M, Yismaw L, Berelie Y, et al.: Health care utilization for common childhood illnesses in rural parts of Ethiopia: evidence from the 2016 Ethiopian demographic and health survey. BMC Public Health. 2019; 19(1): 57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJafari H: Major infectious diseases of children in developing countries: Challenges and opportunities of today and the future. Semin. Pediatr. Infect. Dis. 2004; 15(3): 121â123. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAli R, Shadeed A, Fitian H, et al.: The difficulties experienced during the preparation and administration of oral drugs by parents at home: a cross-sectional study from Palestine. BMC Pediatr. 2020; 20(1): 198. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoztepe H, Ãzdemir H, Karababa Ã, et al.: Administration of oral medication by parents at home. J. Clin. Nurs. 2016; 25(21-22): 3345â3353. Publisher Full Text\n\nBergene E, RÞ T, Steinsbekk A: Strategies parents use to give children oral medicine: a qualitative study of online discussion forums. Scand. J. Prim. Health Care. 2017; 35(2): 221â228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlKaddour NA, Shah RB, Gillani SW, et al.:A cross-sectional survey among parents to report challenges and barriers in the administration of drugs to children. figshare. [Dataset].2022. Publisher Full Text\n\nNarchi H, Elghoudi A, Al Dhaheri K: Barriers and challenges affecting parentsâ use of adrenaline auto-injector in children with anaphylaxis. World J. Clin. Pediatr. 2022; 11: 151â159. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBaguley D, Lim E, Bevan A, et al.: Prescribing for childrenâtaste and palatability affect adherence to antibiotics: a review. Arch. Dis. Child. 2012; 97: 293â297. PubMed Abstract | Publisher Full Text\n\nReis A, Joaquim J: Drug Interaction with Milk and The Relevance of Acidifying/Alkalizing Nature of Food. Clin. Ther. 2015; 37(8): e67âe68. Publisher Full Text\n\nPápai K, Budai M, Ludányi K, et al.: In vitro foodâdrug interaction study: Which milk component has a decreasing effect on the bioavailability of ciprofloxacin? J. Pharm. Biomed. Anal. 2010; 52(1): 37â42. Publisher Full Text\n\nBushra R, Aslam N, Khan AY: Food-drug interactions. Oman Med. J. 2011; 26: 77â83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShehnaz SI, Khan N, Sreedharan J, et al.: Self-medication and related health complaints among expatriate high school students in the United Arab Emirates. Pharm. Pract. 2013; 11(4): 211â218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlbatti T, Alawwad S, Aldueb R, et al.: The self medication use among adolescents aged between 13â18 years old; Prevalence and behavior, Riyadh â Kingdom of Saudi Arabia, from 2014â2015. Int. J. Pediatr. Adolesc. Med. 2017; 4(1): 19â25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRuiz ME: Risks of self-medication practices. Curr. Drug Saf. 2010; 5: 315â323. Publisher Full Text\n\nDu Y, Knopf H: Self-medication among children and adolescents in Germany: results of the National Health Survey for Children and Adolescents (KiGGS). Br. J. Clin. Pharmacol. 2009; 68: 599â608. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGracia-Vásquez SL, González-Barranco P, Camacho-Mora IA, et al.: Medications that should not be crushed. Med. Univ. 2017; 19. : 50â63. Publisher Full Text\n\nWong IC, Ghaleb MA, Franklin BD, et al.: Incidence and nature of dosing errors in paediatric medications. Drug Saf. 2004; 27: 661â670. Publisher Full Text\n\nYin HS, Dreyer BP, Ugboaja DC, et al.: Unit of measurement used and parent medication dosing errors. Pediatrics. 2014; 134(2): e354âe361. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSztajnkrycer M, Bond G: Chronic acetaminophen overdosing in children: risk assessment and management. Curr. Opin. Pediatr. 2001; 13: 177â182. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "157323",
"date": "13 Jan 2023",
"name": "Subish Palaian",
"expertise": [
"Reviewer Expertise Rational use of medicines"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI take this opportunity to thank the Editor for giving me an opportunity to review this manuscript. It is novel research. There is a scope for improvement in the manuscript. Here are my comments:\nTitle: The title should mention âUnited Arab Emirates'\nAbstract: The first line is not properly written. It may be edited as âŠ. inappropriate medicine administration practices. I suggest the authors use the term âmedicineâ instead of âdrugâ. The results should focus on more significant findings.\nIntroduction: This section should clearly specify the reasons for undertaking the study. The study rationale is not clear. The authors are expected to provide more literature evidence to justify the study rationale. A more detailed literature review covering UAE and neighboring countries is needed. There is a scope for adding more information in this section. All possible similar studies should be cited.\n\nMethods: Study design, This study will be conductedâŠâŠ. The term âwill beâ has to be edited. Further, âpublic placesâ is not relevant. Authors can remove it.\n\nResearch tool:\nMore detail information on the preparation, validation, and reliability analysis of the tool is needed. If the tool used is an adopted one, hence permission for the use of the tool and references have to be provided.\n\nStatistical analysis:\nThe version number of SPSS has to be provided. There is a scope for performing more inferential statistical analysis to arrive at more concrete findings based on the p values.\nDiscussion: This section is too short. More literature evidence should be compared with the current findings. The discussion should also focus on the study findings and its significances for future practices. Any potential policy implications based on the study findings have to be explored. Since the study is conducted in the UAE, the discussion must focus on literature and practices from UAE.\nLimitations: A convenient sampling method followed in the research must be added as a limitation.\n\nReferences: Authors are required to provide more recent references wherever possible.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9393",
"date": "21 Mar 2023",
"name": "Syed Wasif Gillani",
"role": "Author Response",
"response": "Response to reviewer  Tittle is changed.  Abstract is updated.  New points were added for the introduction, in addition to references.  Proper grammar in the study design.  Research tool section now explains the permission  Spss version is updated, in this study we are focused on descriptive analysis, but we are planning to conduct data from different parts in UAE and inferential statistics.  Discussion references are added from neighbouring countries.  Limitations: Convenient sampling method was added as a limitation."
}
]
},
{
"id": "157320",
"date": "01 Feb 2023",
"name": "Amjad Khan",
"expertise": [
"Reviewer Expertise Clinical Pharmacy",
"Public Health",
"Clinical Trials"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the invitation to review this study. The authors made a good effort to explore the parents' reported challenges on drug administration among children in UAE. I have a few minor comments on this manuscript.\nAbstract requires revisions on highlighted the important findings only.\n\nWhole manuscript requires copy and language editing for minor grammatical and syntax errors.\n\nProvide limitations of the study specifically related to connivence sampling technique\n\nEach table and graph should have a highlighted summary in the beginning. Also provide abbreviations in the end of each table.\n\nResults section need minor revision with sequence of data according to objectives of the study.\n\nPlease add recent references in the discussion to support the findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9394",
"date": "21 Mar 2023",
"name": "Syed Wasif Gillani",
"role": "Author Response",
"response": "Response to the reviewer: Abstract changes with only highlighted outcomes. Â The manuscript's language and grammar are revised and edited. Â Three study limitations, particularly those related to the concordance sampling technique, were added. Â A summary is already provided, and abbreviations were added. Â The Results section is now written based on the objectives of the study. Â updated references were added."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1431
|
https://f1000research.com/articles/12-311/v1
|
21 Mar 23
|
{
"type": "Research Article",
"title": "Enhancing workplace exercise and physical fitness: university workersâ attitudes",
"authors": [
"Aungkana Boonsem",
"Anan Malarat",
"Aditep Na Phatthalung",
"Anan Malarat",
"Aditep Na Phatthalung"
],
"abstract": "Background: Workplace exercise (WE) has become the focus of a number of studies in Thailand universities as it is believed to prevent numerous chronic diseases. This study aims to illustrate and investigate the behavioral factors related to the physical fitness of university workers and attitudes in work performance.\n\nMethods: We conducted a quasi-experimental study on 93 workers with purposive sampling using the Taro Yamane method, to examine exercise behaviors and knowledge, attitude, and exercise practice. Then, 31 selected participants who were at a moderate level of attitude and exercise practice joined the workplace exercise program for two months. Participantsâ exercise behavior and knowledge, attitude, and exercise practice before and after joining the program were analyzed using an independent t-test. The physical fitness and work attitude were examined. Results: The results show that the university workers who participated in the program had an adjustment in exercise behavior after engaging in WE with a high level of knowledge, attitude (3.14+.18) and exercise practice (3.32+.31). In addition, we found that programs improved physical fitness with a significant increase in resting heart rate and leg strength (p<0.05). A direct relationship between work attitude and physical fitness after adapting intervention were revealed with work attitude in 3.35+.44 high agreement.\n\nConclusion: The results correspond to earlier studies on exercise factors with the increasing exercise behavior level in terms of knowledge, attitude, and exercise practice. The WE is important for university workers. Therefore, the developed WE in the workplace increased the health and work attitude.",
"keywords": [
"workplace exercise",
"exercise behaviors",
"exercise psychology",
"physical activity",
"public health",
"university",
"physical fitness",
"work attitude"
],
"content": "Introduction\n\nIncreasing workplace exercise (WE) has received a lot of attention over the years in learning and sharing health information because during working hours that employees were confronted with health problems.1â3 However, physically active workers are more productive than those who are inactive and overweight. Therefore, poor worker health indicates reduced performance and increased expenses of company. This is the key reason why many worldwide employers have begun focusing on addressing aspects of their workers' health and considering numerous health programs or fitness activities.4â8\n\nIn universities of Bangkok, employees often sit at their desks during working hours without engaging in exercise. Therefore, universities are considering a proper workplace fitness program to reduce indirect health-care costs and promote good health. Workplace exercise encourages individual workers to develop healthy behaviors that then leads to lower costs and increased productivity.9â11 At any age, physical activity is valuable to health. The target of such healthcare inventions is to improve worker fitness and not just simply to reduce the risks. It is evident that a person who is physically fit can perform better at work and withstand workload pressures over-time, and thus be more productive compared to an unfit person.12 This paradigm shift is noticeable across many organizations as they strive to be completely health focused companies.13 This research is useful for universities given the performance impact of physical activity at the workplace and for the promotion of health and wellness among workers. However, understanding the factors on exercise behavior (knowledge, attitude, and exercise practice) related to physical activity among workers is important to establishing an effective WE program. Therefore, the present study aimed to predict the behavioral factors related to physical fitness among university workers and assessed the effectiveness of workplace exercise (WE) in a health promotion program for university workers to improve health and attitudes to work performance.\n\n\nMethods\n\nThis was a prospective, randomized, uncontrolled study, classified as quasi-experimental research using Taro Yamane14 with a single group of workers who used the two-month health promotion program at King Mongkutâs University of Technology Thonburi (KMUTT), Bangkok, Thailand. The periods of recruitment and data collection were done in three months. Then, the exercise behavior, physical fitness and work attitude of participants were determined.\n\nThis study was conducted among employees in KMUTT. We announced the research in the universityâs public relations brochures, and the details of criteria for participation were included. Participants could be anyone who had worked in KMUTT for at least a year, had no health problems and would be able to join the two-month program. The research guidelines and instructions for applying were also included. There were two stages of recruitment (Figure 1). The first stages of recruitment involved the determination of the exercise behavioral factors. After calculation of the sample size by substituting the numbers into the Yamane formula, there was only a sample size of 88 university workers. To obtain reliable data, the researcher increased the sample size to 93 workers out of a total of 753 purposive sampling with any age and mixed gender to evaluate exercise behaviors. In the second stage, healthy workers who had moderate agreement with exercise attitudes and moderate frequency of exercise practice in exercise behavior were selected for the assessment of health promotion program on physical fitness and work attitudes. Therefore, only 31 out of 93 workers were recruited before verifying the work attitude after finishing the program. In addition, they had not previously participated in any programs in six months.\n\nHowever, the exclusion criteria included having any illness before starting the program. No participant withdrew from the study due to illness.\n\nThere were three WE sessions that were created by human resources at KMUTT including 1) Body combat, Aerobic dance, and Yoga; 2) Running and 3) Fitness that were offered to university workers. The details of this program were explained to participants and each took one session and engaged in the program for two months. The sessions were composed of exercises done individually and in pairs for 30 minutes a day for three days a week, after office hours.\n\n\n\n1. Variables studied\n\n1. Independent variables: gender, age\n\n2. Transmission variables: exercise behavior (knowledge, attitudes, and practices) and WE interventions.\n\n3. Dependent variables: Physical fitness (body mass index, resting heart rate, vital capacity, sit and reach, hand grip strength and leg strength) and work attitude.\n\n2. Research tools\n\nThere were two tools used for data collection in this research. The questionnaires measured the exercise behaviors and work attitudes of university workers at King Mongkut's University of Technology, Bangkok, Thailand. In addition, various health measurement tools were used to measure levels of physical fitness and survey the work attitudes of selected workers.\n\nSection 1: A questionnaire on exercise behaviors was determined and reviewed by five experts in behavioral science and sports science in terms of accuracy, suitability, usefulness, and possibility as adapted from a previous article of Boonsem, A.15 The questionnaire has been validated and the reliability is typically assessed using test-retest reliability, inter-rater reliability or internal consistency. Participants were given the questionnaires before and after joining the health promotion program. The questionnaire format consisted of the following aspects.\n\nPart 1: General information: gender and age\n\nPart 2: Exercise behaviors of university workers:\n\n1. Knowledge: A scale was used in the questionnaire to specify the level of knowledge based on the following criteria: High (66.68-100%), Moderate (33.34-66.67%), and Low (0-33.33%).\n\n2. Attitudes: A scale was used in the questionnaire to specify the level of the agreement or disagreement based on the following criteria: High (3.00-4.00) Moderate (2.00-2.99) and Low (1.00-1.99).\n\n3. Exercise practice: A scale was used in the questionnaire to specify the level of the exercise frequency based on the following criteria: High (3.00-4.00) Moderate (2.00-2.99) and Low (1.00-1.99).\n\nSection 2: The tools used to measure physical fitness were based on the Standards of Physical Fitness for the People, as used by the Bureau of Sports Science, the Department of Physical Education and the Ministry of Tourism and Sports, 2019. These consisted of the following five items:16\n\n1. Body Mass Index (BMI) is used to evaluate body proportion by using body weight and height measurements.\n\n2. Pulse rate is used to measure the resting heart rate in the number of heart beats per minute.\n\n3. Volume Lung capacity is used to measure the volume change of the lung between a full inspiration to total lung capacity.\n\n4. Sit and Reach is used to assess weakness in the lower back and rear thighs by bending the knees and body to extend arms forward as far as possible and record the distance.\n\n5. Hand grip strength is used to evaluate the muscle strength and lower arms by the hand grip dynamometer and recording the force in kilograms.\n\n6. Leg strength is used to determine leg strength by dynamometer.\n\nSection 3: A questionnaire on work attitude was determined.17 A scale was used in the questionnaire to specify the level of work attitude based on the following criteria: High (3.00-4.00) Moderate (2.00-2.99) and Low (1.00-1.99)\n\nThe data obtained from the questionnaires and physical fitness were analyzed and divided into the following categories:\n\n1. The data concerning the general background of the subjects were calculated and presented in terms of frequency distribution (n), percentage (%), mean (X¯) and standard deviation (SD).\n\n2. The statistical methods used in the study were on the factors affecting the exercise behavior that influenced the performance of the body (physical fitness) as an independent t-test. The statistical significance was at a level of p< 0.05.\n\nThis research has been reviewed and approved for research ethics committee, School of Liberal Arts, King Mongkutâs University of Technology Thonburi (ID SoLA-EA-2020-1-008). Written informed consent was obtained from participants who were told that their data will be used and published in the consent form.\n\n\nResults\n\nThe gender, age, exercise behaviors and knowledge, attitude, and exercise practice for 93 university workers are shown in Table 1. The overall percentage of knowledge is 88.92±19.08 in high level of knowledge, while the exercise attitude is 2.99 ± 0.34 in moderate agreement of attitude and exercise practice is 2.89 ± 0.65 in moderate frequency of practice. The raw data can be found under Underlying data.42\n\nExercise behavior levels before and after engaging in the exercise program\n\nAfter assessment of the exercise behavior, a total of 31 out of 93 participants with moderate agreement with exercise attitudes and moderate frequency of exercise practice were selected. They consisted of 9 males (29.0%) and 22 females (71.0%), however, most of the workers were female, which justified the fact that a high population of females working in the university have awareness of health. The age of selected workers in the program between 21-30 years consisted of 13 workers (41.9%), 31-40 years were 10 workers (32.3%), 41-50 years were 7 workers (22.6%) and 51-60 years for 1 worker (3.2%).\n\nIn terms of clarifying the exercise behavior after the engagement program, the results of t-test analyses comparing exercise behaviors in terms of knowledge, attitudes, and exercise practice before and after the engagement program are shown in Table 2. The data showed a significant increase in attitude and exercise practice, with a statistical significance at a level of 0.05. The adjustment on exercise attitude from 2.96 to 3.14 out of 4.00, indicated moderate to high level of attitude to exercise as well as the change from 2.96 to 3.32 in exercise practice, from the moderate level to the high level in terms of exercise practice. However, increases in knowledge at a high level were shown to have no effect after engaging in the program.\n\nEffects of programs on physical fitness\n\nThe analysis of the paired t-test was computed and the results of programs affecting physical fitness for university workers was presented in Table 3. A comparison of physical fitness before and after engaging in the program indicated body mass index, vital capacity, sit and reach and hand grid strength had no significantt change. However, there was a significant increase in resting heart rate and leg strength with a statistical significance of.05 as shown in Table 3. In addition, the results of WE interventions showed that resting heart rate, vital capacity, sit and reach, hand grip strength and leg strength demonstrated that the average level of physical fitness improved with a decrease in resting heart rate and increased vital capacity, sit and reach, hand grip strength and leg strength with no appearance in BMI.\n\nEffect of the program on attitudes to work\n\nAfter an analysis of data after engaging in program on work performance, the results\n\nfound that the overall work attitude levels of university workers were 3.35±.44 with high agreement terms of work attitude, as shown in Table 4.\n\n\nDiscussion\n\nThe purpose of this study was to evaluate the behavioral factors related to physical fitness among university workers and assess the effectiveness of workplace exercise (WE) in a health promotion program construct for university workers and its influence on health-related and work-related attitudes. In the area of occupational health related to WE, this area study occurred due to the need to launch a worker health-promotion used in various locations.18,19\n\nTherefore, in this regard, the behavior of the outcomes studied after two months of the workplace exercise program were observed, where each employee has the measurement compared in the initial and final moments and then to illustrate and investigate the importance of exercise behavior, physical fitness, and worker attitudes. To understand better why adult individuals do not engage in exercise is important to professionals who develop behavior-changing interventions. Firstly, the present study was the identification of the factors for exercise behavior among university employees by reinforcing and developing the performance of the WE program at universities in Thailand. The results of exercise behavior showed that age and gender had no effect on level of knowledge among university workers at a high level. However, the results of moderate attitude levels and exercise practice agreed with previous studies on theoretical assumptions of the health action process approach.20 In terms of the theory of changing exercise behavior based on the transtheoretical model, the exercise behavior is mentioned above and inspired an interesting study.\n\nThe promotion of exercise behavior is a potential solution for physical inactivity problems among university workers. The factors understanding impacts on university workers exercise behavior is crucial for university exercise promoting intervention. We argued that general exercise factors cannot comprehensively clarify exercise behavior of university workers in a specific university context.21 Therefore, this study determined the factors of 31 selected university workers with moderate attitude levels and exercise practice after engaging in the program. The current results confirmed and extended earlier studies on exercise factors with the increasing exercise behavior level in terms of knowledge, attitude, and exercise practice. In addition, we also confirmed that the exercise factors in the workplace are enjoyment, perceived health, competence, and available time. In accordance with Dishman et al. who recommended that strong factors of exercise participation are behavioral skills, perception of good health, self-motivation, and leisure time.22 We also established that influence of friends and colleagues are also determinants for the exercise behavior of university workers, as shown in earlier findings where people in both home and work environments demonstrated another robust correlation of exercise behavior.23,24 Besides that, there were three factors (work pace, company policy, and work burden) that extended the exercise factor in studies in a workplace and in a site-specific manner. The factor of work environment also needs to be considered as an important factor in workplace exercise behavior promoting programs. In addition, this finding is supported by several previous studies, in which individuals who perceived more barriers to the exercise were less likely to engage in this behavior.25,26 Pender et al. focused on a perceived barrier as an essential mediator of the motivational readiness of individuals to develop healthier behavior.27 In other studies, perceived barriers predicted physical activity behavior.28,29 In order to promote WE, it is necessary to solve the perceived barriers, which can be defined as predictive. This study also showed the valuable effects of commitment to action planning and self-efficacy. The previous evidence demonstrated the relationship between a plan of action and maintaining self-efficacy on one hand, and exercise behavior on the other.30 In Laffrey, indicating Bandura, it was noted that the perception of self-efficacy influenced the emotional arousal, thoughts, and actions of the individual, and the higher the level perceived efficacy for a behavior, it is more likely that they will accomplish such behavior.31 Moreover, in accordance with our study, numerous studies have shown that perceived self-efficacy has been the best predictor variable for actual exercise activity.32,33 Therefore, strategies for enhancing efficacy in practice through motivational counseling interviews and focus group discussions for strengthening self-efficacy, could lead to more effective health promotion programs for university employees and should be considered in developed interventions.34\n\nAmong the outcomes studied in terms of the relationship between exercise behaviors and physical fitness, significant differences are noted between the initial and final evaluations in terms of a resting heart rate (p<0.005) and leg strength (p<0.01). A high level of exercise behavior in terms of knowledge, attitudes and exercise among university workers effected on physical fitness in terms of resting heart rate, vital capacity, sit and reach, hand grip strength and leg strength. However, there was no effect on body mass index, because the factors of diet had also affected their physical fitness. However, the studies that researched the effects of WE on body mass, muscle force and flexibility have found benefits.35,36 The exercise behavior of university workers were stimulated, and it encouraged them to develop strong muscles, flexibility, and endurance of the circulatory system to adjust the exercise behavior of university workers with the aim of developing a healthy body. In terms of BMI, the fat reduction affected their body mass index after exercise routines and regarding consumption behavior.37 A balanced diet, controlled portions and the amount of food is also an important factor in determining BMI.38 Workers who engaged in the program had a low resting heart rate confirming the effectiveness of the WE program on the performance of the heart system and blood circulation.\n\nAlthough leg strength improved after the program, the flexibility, strength and endurance of the leg muscles in sit and reach need to develop physically continuous support of a body motion on a continuous basis and causing physiological adaptations tends to increase muscle strength and results in smoother movements.39 In order to increase hand grip strength, the training required an increase in the strength and endurance of the arm muscles, but the development machine exercise focused on gross motor skills and the improvement of arm muscle abilities.40 Therefore, the data indicated that exercise behaviors were related to physical fitness as well as the ability to manage their environment, mental states, perception, and influence in determining the abilities of the individuals. The habit of exercise routines in behavioral practice increased their knowledge and attitude to fitness as importance and regarding practice on a daily basis. In addition, the results demonstrated university workers who are in good physical fitness have a high level of exercise behavior.\n\nThe study stated that subjective health would mediate the physical exercise and work attitude relationship. The results of the study identified a positive mediation of subjective health to work attitudes. The participants who engaged in physical exercise experience increased their feelings of well-being.41 The health status of employees directly influenced their on-the-job performance and improved employee well-being, which results in a more productive workforce.17\n\nIn terms of the gap in the relationship between workplace exercise program in physical fitness to the attitudes or university workers in Bangkok, Thailand, this study suggested that some physical activity programs are effective beyond direct health benefits. The design of an intervention benefits from a multidisciplinary team with different perspectives to set of goals. Such work has great potential to make a large impact on the health and well-being of university workers by intervening and making highly beneficial lifestyle changes.\n\n\nConclusion\n\nThe achieved outcomes indicated that physical exercise levels are positively impacted by fitness and workerâs productivity. The measured results improved job attitudes through enrollment in the exercise program by employees. In addition, this study showed that there is a direct relationship between work attitude and physical fitness after adapting exercise behaviors by WE program. In general, workers who participated in WE interventions had more self-confidence and concentration at work, and the physical activity can reduce stress and depression while under pressure in the workplace. However, the limitation of this research is the data on health problems because workplace exercise increases health of workers and reduces the cost of treating illness. Furthermore, introducing a workplace policy to provide university workers with subsidized gym/recreation memberships or fitness counseling are an effective way to increase and support worker physical activity and to reduce barriers to physical exercise, such as cost.",
"appendix": "Data availability\n\nFigshare: Raw data. https://doi.org/10.6084/m9.figshare.22122374.v3. 42\n\nThis project contains the following underlying data:\n\n- raw.data.xlsx [data on exercise behaviors and physical fitness]\n\nThis project contains the following extended data:\n\n- Questionnaire\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors would like to acknowledge school of Liberal Arts, King Mongkut's University of Technology, Thonburi, Bangkok, Thailand for all their support.\n\n\nReferences\n\nKelly F: Guidelines on improving the physical fitness of employees. WHO Regional Office for Europe; 2000.\n\nStraker L, Mathiassen SE: Increased physical workloads in modern workâA necessity for better health and performance? Ergonomics. 2009; 52: 1215â1225. PubMed Abstract | Publisher Full Text\n\nBize R, Plotnikoff RC: The relationship between a short measure of health status and physical activity in a workplace population. Psychol. Health Med. 2008; 14: 53â61. PubMed Abstract | Publisher Full Text\n\nJaner G, Sala M, Kogevinas M: Health promotion traits at worksites and risk factors for cancer. Scand. J. Work Environ. Health. 2002; 28(3): 141â157. PubMed Abstract | Publisher Full Text\n\nEngbers LH, van Poppel MN , Paw MJ, et al.: Worksite health promotion programs with environmental changes: a systematic review. Am. J. Prev. Med. 2005; 29(1): 61â70. Publisher Full Text\n\nShephard R: Worksite fitness and exercise programs: a review of methodology and health impact. Am. J. Health Promot. 1996; 10: 436â452. PubMed Abstract | Publisher Full Text\n\nMohammed M, Naji FL: Benefits of exercise training for computer-based staff: a meta-analysis. Int. J. Kinesiol. Sports Sci. 2017; 5: 16â23. Publisher Full Text\n\nBrand R, Schlicht W, Grossmann K, et al.: Effects of a physical exercise intervention on employees' perceptions of quality of life: a randomized controlled trial. Soz. Praventivmed. 2006; 51(1): 14â23. PubMed Abstract | Publisher Full Text\n\nAldana SG, Jacobson BH, Harris CJ, et al.: Influence of a mobile worksite health promotion program on health care costs. Am. J. Prev. Med. 1993; 9(6): 378â383. PubMed Abstract | Publisher Full Text\n\nBernaards CM, Proper KI, Hildebrandt VH: Physical activity, cardiorespiratory fitness and body mass index in relationship to work productivity and sickness absence in computer workers with preexisting neck and upper limb symptoms. J. Occup. Environ. Med. 2007; 49: 633â640. PubMed Abstract | Publisher Full Text\n\nMills PR, Kessler RC, Cooper J, et al.: Impact of a health promotion program on employee health risks and work productivity. Am. J. Health Promot. 2007; 22: 45â53. Publisher Full Text\n\nSharifzadeh M: Does fitness and exercises increase productivity? Assessing health, fitness and productivity relationship. Am. J. Manag. 2013; 13: 32â52.\n\nProper KI, Koning M, Van der Beek AJ, et al.: The effectiveness of worksite physical activity programs on physical activity, physical fitness, and health. Clin. J. Sport Med. 2003; 13(2): 106â117. Publisher Full Text\n\nYamane T: Statistics: An introductory analysis. 3rd ed.New York: Harper and Row Publications; 1973.\n\nBoonsem A, Malarat A, Prachanban S: Relationship between the factors affecting exercise behaviour and physical fitness among university students. J. Phys. Educ. 2021; 32: e3277. Publisher Full Text\n\nMinistry of Tourism and sports: Test and benchmark for physical fitness of people aged 19-59 years. Bangkok: Bureau of Sports science; 2019. [cited on 15 June 2022]. Reference Source\n\nDrannan J: The Relationship Between Physical Exercise and Job Performance: The Mediating Effects of Subjective Health and Good Mood. Arabian J. Bus. Manag. Review. 2016; 6: 6.\n\nDishman RK, DeJoy DM, Wilson MG, et al.: Move to Improve: a randomized workplace trial to increase physical activity. Am. J. Prev. Med. 2009; 36(2): 133â141. Publisher Full Text\n\nGrande AJ, Silva V, Parra SA: Effectiveness of exercise at workplace in physical fitness: uncontrolled randomized study. einstein. 2014; 12(1): 55â60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcEachan RR, Conner M, Taylor NJ, et al.: Prospective prediction of health-related behaviors with the theory of planned behaviors: a meta-analysis. Health Psychol. Rev. 2011; 5: 97â144. Publisher Full Text\n\nZhang T, Ham J, Ren X: Why Exercise at Work: Development of the Office Exercise Behavior Determinants Scale. Int. J. Environ. Res. Public Health. 2021; 18: 2736. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDishman RK, Sallis JF, Orenstein DR: The Determinants of Physical Activity and Exercise. Public Health Rep. 1985; 100: 158â171. PubMed Abstract | Free Full Text\n\nHovell M, Sallis J, Hofstetter R, et al.: Identification of correlates of physical activity among Latino adults. J. Community Health. 1991; 16: 23â36. PubMed Abstract | Publisher Full Text\n\nAittasalo M, Miilunpalo S, Suni J: The effectiveness of physical activity counseling in a work-site setting. A randomized, controlled trial. Patient Educ. Couns. 2004; 55(2): 193â202. Publisher Full Text\n\nShin YH, Hur HK, Pender NJ, et al.: Exercise self-efficacy, exercise benefits and barriers, and commitment to a plan for exercise among Korean women with osteoporosis and osteoarthritis. Int. J. Nurs. Stud. 2006; 43: 3â10. PubMed Abstract | Publisher Full Text\n\nJustine M, Azizan A, Hassan V, et al.: Barriers to participation in physical activity and exercise among middle-aged and elderly individuals. Singap. Med. J. 2013; 54: 581â586. Publisher Full Text\n\nPender NJ, Murdaugh CL, Parsons MA: Health Promotion in Nursing Practice. Boston, MA: Pearson; 2015.\n\nChenary R, Noroozi A, Tavafian SS, et al.: Effective factors on health-promoting lifestyle among Iranian chemical veterans in 2014 based on health promotion model: a path analysis. Iran. Red Crescent Med. J. 2016; 18: e33467. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlaviani M, Khosravan S, Alami A, et al.: The effect of a multi-strategy program on developing social behaviors based on penderâs health promotion model to prevent loneliness of old women referred to gonabad urban health centers. Int. J. Community Based Nurs. Midwifery. 2015; 3: 132â134. PubMed Abstract | Free Full Text\n\nSniehotta FF, Scholz U, Schwarzer R, et al.: Long-term effects of two psychological interventions on physical exercise and self-regulation following coronary rehabilitation. Int. J. Behav. Med. 2005; 12: 244â255. PubMed Abstract | Publisher Full Text\n\nLaffrey SC: Physical activity among older Mexican American women. Res. Nurs. Health. 2000; 23: 383â392. Publisher Full Text\n\nWu TY, Pender N: Determinants of physical activity among Taiwanese adolescents: an application of the health promotion model. Res. Nurs. Health. 2002; 25: 25â36. PubMed Abstract | Publisher Full Text\n\nDelshad MH, Tavafian SS, Kazemnejad A: Factors Predicting the Stretching Exercise Behaviors of Office Employees Working in the Shahid Beheshti University of Medical Sciences in Tehran, Iran. Rev. Invest. Clin. 2019; 71: 178â185. Publisher Full Text\n\nPender NJ, Murdaugh CL, Parsons MA: Health Promotion in Nursing Practice. 4th ed.Upper Saddle River, New Jersey: Prentice Hall; 2002.\n\nFuruki K, Honda S, Jahng D, et al.: The effects of a health promotion program on body mass index. J. Occup. Health. 1999; 41: 19â26. Publisher Full Text\n\nCaban-Martinez AJ, Lowe KA, Herrick R, et al.: Construction workers working in musculoskeletal pain and engaging in leisure-time physical activity: findings from a mixed-methods pilot study. Am. J. Ind. Med. 2014; 57: 819â825. PubMed Abstract | Publisher Full Text | Free Full Text\n\nButler CE, Ruth Clark B, Burlis TL, et al.: Physical activity for campus employees: a university worksite wellness program. J. Phys. Act. Health. 2015; 12(4): 470â476. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang JH, Huang SL, Li RH, et al.: Effects of nutrition and exercise health behaviors on predicted risk of cardiovascular disease among workers with different body mass index levels. Int. J. Environ. Res. Public Health. 2014; 11: 4664â4675. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCorder K, Ogilvie D, Van Sluijs EM: Invited commentary: Physical activity over the life courseâwhose behavior changes, when, and why? Am. J. Epidemiol. 2009; 170: 1078â1081. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmolander J, Sörensen L, Pekkonen M, et al.: Muscle performance, work ability and physical functioning in middle-aged men. Occup. Med. 2010; 60: 78â80. PubMed Abstract | Publisher Full Text\n\nEtemadi M, Shameli K, Hassan NA, et al.: A Review of the Importance of Physical Fitness to Company Performance and Productivity. Am. J. Appl. Sci. 2016; 13(11): 1104â1118. Publisher Full Text\n\nBoonsem A, Malarat A, Na Phatthalung A: Raw data. Dataset. figshare. 2023. Publisher Full Text"
}
|
[
{
"id": "173839",
"date": "23 Jun 2023",
"name": "Xiaofen D. Keating",
"expertise": [
"Reviewer Expertise fitness testing",
"health-related behavior change",
"participants in higher education"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI fully understand how difficult to conduct an experimental study on changing people's behaviors and attitudes. However, this study has nothing new and is not guided by any theory. The literature review was not adequately done in the introduction section. There is a lack of evidence concerning the instruments used to measure the targeted variables such as knowledge and attitudes. The fatal flaws in the research design cannot be overcome by using different statistical analyses.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-311
|
https://f1000research.com/articles/12-309/v1
|
21 Mar 23
|
{
"type": "Study Protocol",
"title": "State-of-the-art of miliary tuberculosis in children: protocol for a scoping review of the literature",
"authors": [
"Danilo Buonsenso",
"Francesco Mariani",
"Rosa Morello",
"Rinn Song",
"Francesco Mariani",
"Rosa Morello",
"Rinn Song"
],
"abstract": "Background: Miliary Tuberculosis (TB) is a rare manifestation of TB described in both adults and children. Pediatirc reports are rare and therefore evidence about diagnostics and management is scarce. Methods: We will start our research in March 2023 in the following bibliographic databases: PubMed, EMBASE, Cochrane and SCOPUS. The main review question will be âWhat are the main clinical characteristics and outcomes of pediatric miliary TB reported in the literature?â We will include randomized and non-randomized controlled trials, prospective and retrospective observational studies, performed on children and adolescents (younger than 18 years), hospitalized or not, with a confirmed diagnosis of miliary TB. To report our findings, we will follow Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist. Dissemination: The findings of this review will be published in international peer-reviewed journals and presented in pediatric infectious diseases national and international conferences.",
"keywords": [
"miliary TB",
"tuberculosis",
"children"
],
"content": "Introduction\n\nMiliary tuberculosis (TB) is a rare and often fatal (if untreated) form of TB characterized by massive lymphohematogenous dissemination from a Mycobacterium tuberculosis-laden focus.1 Historically, the term was coined by John Jacob Manget when he described the anatomical-pathological findings in certain TB patients who had numerous small lesions which resembled millet seeds in size and appearance.2\n\nAlthough miliary TB has been formally described three centuries ago, several uncertainties remain around this topic. First, several publications confound miliary and disseminated TB or use these terms interchangeably.1,3 However, although the pathogenesis of these two manifestations is similar due to hematogenous spread of the bacilli, the anatomical-pathological findings are different, with the âmiliary patternâ being documented only in some of the patients with multiorgan TB involvement. Therefore, current agreement for the diagnosis of miliary TB is presence of a diffuse miliary infiltrate on chest radiograph or high-resolution computed tomography (CT) scan, or evidence of miliary tubercles in multiple organs at laparoscopy, open surgery, or autopsy.1 However, such definition has intrinsic limitations for the recognition of miliary TB in children, as invasive procedures for anatomical-pathological diagnosis are less frequently performed in children, and CT scan (and sometimes even X-ray) is rarely available in low-to-middle income countries where TB incidence is highest. For these reasons, the current understanding of miliary TB in children is still limited.\n\nTwo recent reviews, published in 2006 and 2016,1,3 analyzed together adults and children and highlighted current knowledge gaps in clinical recognition, diagnosis, optimal treatment and outcomes. Therefore, a systematic approach in collecting comprehensive information from pediatric miliary TB literature has never been performed. Importantly, since the publication of these reviews, the TB diagnostic field has evolved significantly, with the advent of interferon-gamma release assays (IGRAs),4 and the wide-spread availability of molecular (PCR-based) TB tests.5 To date the published literature on the diagnostic performance of current commercially available PCR-based TB assays in children with miliary TB remains limited to a single study that showed lower sensitivity in children with miliary TB, but the assessment of miliary TB was not a primary aim of the study.4\n\nIn addition, there is lack of data on treatments, complications and outcomes on children with miliary TB. Older reports from low-resource settings suggested that miliary TB is associated with a mortality of 30-50%,1,3 however most of these reports were published before 2000 or only reported data from resource-limited settings, where high-level intensive care support is less available. Importantly, even in high income countries intensive care support has significantly improved and may have positively affected outcomes of children with miliary TB.\n\nGiven the mentioned gaps, we will perform a scoping review aiming to summarize the current literature on the main characteristics of pediatric miliary TB covering epidemiology and clinical characteristics, diagnostic and therapeutical options and outcomes. We decided to propose a scoping review because of the need of examining a wide area of knowledge to identify knowledge and research gaps and to provide indicators of topics for a subsequent systematic review. To ascertain that no such reviews were available, a search was performed on MEDLINE, the Cochrane Database of Systematic Reviews, JBI Evidence Synthesis and PROSPERO on December 15th, 2022.\n\n\nReview questions\n\nWhat are the main clinical characteristics and outcomes of pediatric miliary TB reported in the literature?\n\nThis review will investigate these sub-questions:\n\n1. What is known about the epidemiology of pediatric miliary TB?\n\n2. What is known about the diagnostic accuracy of microbiological tests in children with miliary tuberculosis?\n\n3. What is known about the diagnostic accuracy of immunological tests in children with miliary tuberculosis?\n\n4. Which are the main therapeutical options (e.g. number and length of treatment with anti-mycobacterial agents and use of steroids or other adjunctive therapeutics) in the treatment of pediatric miliary TB?\n\n5. What outcomes are reported in the literature on pediatric miliary TB?\n\n\nInclusion criteria\n\nIn this review will be included studies performed on pediatric patients younger than 19 years with a diagnosis of miliary TB according to the currently agreed definitions1,3\n\nWe will focus on pediatric miliary tuberculosis in all its aspects.\n\nNo limitation of care setting will be included. Articles will be considered for inclusion in this review independently to the care setting of children so we will consider both inpatients and outpatients.\n\nThis review will include both randomized controlled trials and non-randomized controlled trials. All the type of observational studies, prospective and retrospective (including case-control, cohort and cross-sectional studies, small case series or single case reports) will be included.\n\n\nMethods\n\nWe will systematically search the following bibliographic databases up to the 31st of January 2022: the Cochrane Library (Cochrane database of Systematic Reviews, Cochrane Central Register of Controlled Trials Central), MEDLINE, Web of Science and SCOPUS.\n\nThere will be no date restrictions, except inclusion of papers written in English. The search strategy will include the following word: âpediatricâ and âmiliary tuberculosisâ (patients younger than 18 years of age will be considered as children or pediatrics). The search strategy for MEDLINE is available as Extended data6; the terms used for this search will be adapted for use with other bibliographic databases.\n\nWe will also conduct a web search, via Google, to find gray literature and unpublished material.\n\nIf the final analysis were to be performed 6 months after the bibliographic search, the search string will be launched again to evaluate the presence of new studies to be included in the work.\n\nWe will export studies in Rayyan. A first screen to exclude duplicates will be performed by one author.\n\nTitles and/or abstracts of retrieved studies will be screened independently by two reviewers to identify those that could be included in the review. Full texts of potentially eligible studies will subsequently be retrieved and independently assessed for eligibility by two blinded reviewers. Disagreement about studiesâ elegibility will be resolved through discussion and, in case of further disagreement, by discussion with a third reviewer.\n\nAll studies that will not meet the inclusion criteria will be excluded and a figure with the reason why those studies were excluded will be inserted in the final manuscript.\n\nThe results of the search will be reported in the PRISMA flow diagram.\n\nTwo blinded reviewers will extract data independently on a different excel file. Any disagreement will be identified and resolved as previously mentioned.\n\nAn Excel file will be used to storage data; extracted information will include:\n\n1. Study general features: title, author, year of publication, type of study, number of patients included in the study, geographical area where the study has been performed\n\n2. Participant features: sample size of each group, nationality, age, socio-economic status\n\n3. Main presenting signs and symptoms: fever (including days), cough (including days), weight loss, vomiting, diarrhea, rashes, neurological symptoms, eye symptoms and fundus oculi, others\n\n4. Main imaging findings: type of lung involvement at chest X-Ray and/or CT scan, type of CNS involvement on CT scan or Magnetic Resonance Imaging (MRI), type of bone or other organ involvement evaluated by ultrasound or CT scan MRI of other organs)\n\n5. TB localizations (e.g., lung, central nervous system, other organs), highlighting in which of these organs a miliary involvement was documented\n\n6. Anatomical-pathological data, where available\n\n7. Sensitivity (and specificity, if available) of microbiological tests (direct microscopy, culture or molecular diagnostics) on different specimens (e.g., gastric lavage, sputum, stools, cerebrospinal fluid, other)\n\n8. Sensitivity (and specificity, if available) of immunological tests (tuberculin skin test, interferon gamma releasing assays of different generations (e.g., T-SPOT, QFT-Gold, QFT-Plus)\n\n9. Antimicrobial treatments provided and length of therapy\n\n10. Other treatments provided (e.g., steroids or other immunomodulatory medications)\n\n11. Outcomes (death, survival; survival with or without sequelae; type of sequelae)\n\nTo report our findings, we will follow Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist.\n\nWe will produce a narrative synthesis of the findings from the studies included in the review describing the results we have obtained and provide our opinion on their interpretation. More specifically, we will summarize the evidence using a descriptive summary that includes the characteristics of included studies (number of studies, study design, year of publication, characteristics of the study populations, and countries where studies were conducted).\n\nWe will also use tables and charts to summarize the most important clinical, diagnostics, treatment and outcome data. We will provide five different summary tables or figures summarizing main data about clinical presentation, imaging characteristics, diagnostics (microbiology and immunology) information, treatments provided and outcomes. We will provide both descriptive findings and, were possible, sensitivity and specificity for diagnostics according to the study designs.\n\nBy doing so, we will be able to provide the most up-to-date and comprehensive platform of information about what is currently known of pediatric miliary TB and, therefore, guide future research projects to fill current gaps.\n\nTwo blinded reviewers have started abstract screening of February 2023.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\nFigshare: Appendix 1. https://doi.org/10.6084/m9.figshare.22154999.v1. 6\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgments\n\nWe are grateful to Matthew Henry (Outreach Librarian, Acute General Medicine, Allied Health Therapies, BRC, Gastroenterology, Paediatrics, Pharmacy, Bodleian Health Care Libraries, University of Oxford) for its support in implementation of the search strategy.\n\n\nReferences\n\nSharma SK, Mohan A: Miliary Tuberculosis. Microbiol. Spectr. 2017 Mar; 5: 2. PubMed Abstract | Publisher Full Text\n\nManget JJ: Observatio XLVII. Sepulchretum sive anatomica practica. London, England: Cramer and Perachon; vol. 1. . 1700.\n\nMert A, Ozaras R: A terminological controversy: do disseminated and miliary tuberculosis mean the same? Respiration. 2005 Jan-Feb; 72(1): 113. PubMed Abstract | Publisher Full Text\n\nBuonsenso D, Noguera-Julian A, Moroni R, et al.: Performance of QuantiFERON-TB Gold Plus assays in paediatric tuberculosis: a multicentre PTBNET study. Thorax. 2022 Oct 25; 78: 288â296. PubMed Abstract | Publisher Full Text\n\nAguilera-Alonso D, SolÃs-GarcÃa G, Noguera-Julian A, et al.: Accuracy of Xpert Ultra for the diagnosis of paediatric tuberculosis in a low TB burden country: a prospective multicentre study. Thorax. 2022 Oct; 77(10): 1023â1029. PubMed Abstract | Publisher Full Text\n\nMariani F: Appendix 1. figshare. Figure.2023. Publisher Full Text"
}
|
[
{
"id": "167136",
"date": "12 Apr 2023",
"name": "Antoni Soriano Arandes",
"expertise": [
"Reviewer Expertise Pediatric TB and Ped Infect Diseases"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear authors,\nThank you very much for submitting this scoping review. The methods you use for the review are appropriate and the review of this issue is necessary due to the lack of scientific evidence as you stated in your introduction.\nThe results of this review will be crucial to distinguish outcomes of miliary TB in paediatric population from outcomes reported from other TB forms (disseminated) not included in this scope.\nNothing else to add or comment.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-309
|
https://f1000research.com/articles/12-308/v1
|
21 Mar 23
|
{
"type": "Data Note",
"title": "The identification of high-performing antibodies for transmembrane protein 106B (TMEM106B) for use in Western blot, immunoprecipitation, and immunofluorescence",
"authors": [
"Riham Ayoubi",
"Maryam Fotouhi",
"Kathleen Southern",
"Ritika Bhajiawala",
"Rebeka Fanti",
"Panagiotis Prinos",
"Peter S. McPherson",
"Carl Laflamme",
"NeuroSGC/YCharOS/EDDU collaborative group",
"ABIF Consortium",
"Riham Ayoubi",
"Maryam Fotouhi",
"Kathleen Southern",
"Ritika Bhajiawala",
"Rebeka Fanti",
"Panagiotis Prinos",
"Peter S. McPherson"
],
"abstract": "Transmembrane protein 106B (TMEM106B), a protein that is localized to the lysosome, is genetically linked to many neurodegenerative diseases and forms fibrils in diseased brains. The reproducibility of TMEM106B research would be enhanced if the community had access to well-characterized anti-TMEM106B antibodies. In this study, we characterized six commercially available TMEM106B antibodies for their performance in Western blot, immunoprecipitation, and immunofluorescence, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.",
"keywords": [
"Uniprot ID Q9NUM4",
"TMEM106B",
"Transmembrane protein 106B",
"antibody characterization",
"antibody validation",
"Western Blot",
"immunoprecipitation",
"immunofluorescence"
],
"content": "Introduction\n\nTransmembrane protein 106B (TMEM106B) is a genetic risk variant for many neurodegenerative diseases. The presence of the TMEM106B major risk allele, rs1990622, is suspected to be a risk factor and disease modifier for Frontotemporal Dementia (FTD), with few studies investigating its potential role in Amyotrophic Lateral Sclerosis (ALS) pathogenesis.1â5\n\nTMEM106B is a transmembrane endosomal and lysosomal glycoprotein. The protein has garnered interest lately, with the discovery that a 135 amino acid portion of the protein from its luminal C-terminal domain forms fibrils in the brains of patients with frontotemporal lobar degeneration, progressive supranuclear palsy, and dementia with Lewy bodies.6,7\n\nThe roles of TMEM106B fibrils in normal lysosomal function or disease pathogenesis are not known, nor is the mechanism by which the protein is proteolyzed, or forms fibrils.7 Mechanistic studies would be greatly facilitated with the availability of high-quality validated antibodies. Here, we compared the performance of a range of commercially available antibodies for TMEM106B and characterized several high-quality antibodies for Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of TMEM106B properties and function.\n\n\nResults and discussion\n\nOur standard protocol involved comparing readouts from wild-type (WT) and knockout (KO) cells.8,9 The first step was to identify a cell line(s) that expresses sufficient levels of TMEM106B to generate a measurable signal. To this end, we examined the DepMap transcriptomics databases to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million âTPMâ +1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HAP1 cells expressed the TMEM106B at RNA levels above the average range of cancer cells analyzed. The parental and KO HAP1 cell lines were obtained from Horizon Discovery (Table 1).\n\nFor Western blot experiments, we resolved proteins from WT and TMEM106B KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).\n\nLysates of HAP1 (WT and TMEM106B KO) were prepared and 15 ÎŒg of protein were processed for Western Blot with the indicated TMEM106B antibodies. The Ponceau stained transfers of each blot are presented are shown equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 93334** and 60333-1-Ig*, which were titrated to 1/500 and 1/2000, respectively, as the signal was too weak when following the supplierâs recommendations. Antibody dilution used: ab244516 at 1/500, A20165 at 1/2000, 93334** at 1/500, 60333-1-lg* at 1/2000, PA5-34353 at 1/500, and PA5-63558 at 1/200.Predicted band size: 31 kDa. *= monoclonal antibody, **= recombinant antibody.\n\nFor immunoprecipitation experiments, we used the antibodies to immunopurify TMEM106B from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the TMEM106B protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).\n\nHAP1 lysates were prepared, and IP was performed using 2.0 ÎŒg of the indicated TMEM106B antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for Western Blot with the indicated TMEM106B antibody. For Western Blot, 93334** was used at 1/500. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=2% starting material; UB=2% unbound fraction; IP=immunoprecipitate; HC=antibody heavy chain. *= monoclonal antibody, **= recombinant antibody.\n\nFor immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.10 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).\n\nHAP1 WT and TMEM106B KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with a glass bottom. Cells were stained with the indicated TMEM106B antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels were performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When the concentration was not indicated by the supplier, we tested antibodies at 1/1000 or 1/2000. At this concentration, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab244516 at 1/100, A20165 at 1/2000, 93334** at 1/100, 60333-1-lg* at 1/2000, PA5-34353 at 1/1000, and PA5-63558 at 1/100. Bars = 10 ÎŒm. *= monoclonal antibody, **= recombinant antibody.\n\nIn conclusion, we have screened many TMEM106B commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions.\n\n\nMethods\n\nAll TMEM106B antibodies are listed in Table 2 together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.11 Peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies are from Thermo Fisher Scientific (cat. number 62-6520 and 65-6120). Alexa-555 conjugated secondary goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424)\n\n* Monoclonal antibody.\n\n** Recombinant antibody.\n\n1 Refer to RRID recently added to the Antibody Registry (on January 2023), they will be available on the Registry website in coming weeks.\n\nBoth HAP1 WT and TMEM106B KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.12 Cells were cultured in DMEM high glucose (GE Healthcare, cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent, cat. number 609065), 100 IU penicillin and 100 ÎŒg/mL streptomycin (Wisent, cat. number 450201).\n\nWestern blot experiments were performed as described in our standard operating procedure.13 HAP1 WT and TMEM106B KO were collected in RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) from Thermo Fisher Scientific (cat number 0089901), supplemented with 1x protease inhibitor cocktail mix from MilliporeSigma (cat. number 78429). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 x g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.\n\nWestern blots were performed with pre-cast mini 4-15% gradient polyacrylamide gels from Bio-Rad (cat. number 4561084) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western blots. Blots were blocked with 5% milk for 1 h, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095) in TBS with 0,1% Tween 20 (TBST) (Cell Signaling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 ÎŒg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes are incubated with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41).\n\nImmunoprecipitation was performed as described in our standard operating procedure.14 Antibody-bead conjugates were prepared by adding 2 ÎŒg of antibody to 500 ÎŒL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a 1.5 mL microcentrifuge tube together with 30 ÎŒL of Dynabeads protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked ~2 hours at 4°C followed by several washes to remove unbound antibodies.\n\nHAP1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) from Thermo Fisher Scientific (cat. number 87788), supplemented with protease inhibitor from MilliporeSigma (cat. number P8340). Lysates were rocked for 30 min at 4°C and spun at 110,000 x g for 15 min at 4°C. 0.5 mL aliquots at 2.0 mg/mL of lysate were incubated with an antibody-bead conjugate for ~2 hrs at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP lysis buffer and processed for SDS-PAGE and Western blot on a pre-cast mini 4-15% polyacrylamide gel. Prot-A:HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a dilution of 0.4 ÎŒg/mL.\n\nImmunofluorescence was performed as described in our standard operating procedure.10 HAP1 WT and TMEM106B KO were labelled with CellTracker green (Thermo Fisher Scientific, cat. number C2925) or CellTracker deep red (Thermo Fisher Scientific, cat. number C34565) fluorescence dye, respectively. The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. number D3571) fluorescent stain. WT and KO cells were plated in 96 well glass plates (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37oC, 5% CO2. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10mL) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed three times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS containing 5% BSA, 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary TMEM106B antibodies overnight at 4°C. Cells were then washed 3 à 10 min with IF buffer and incubated with the corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 ÎŒg/mL for 1 hr at room temperature with DAPI. Cells were washed 3 à 10 min with IF buffer and once with PBS.\n\nImages were acquired on an ImageXpress micro widefield high-content microscopy system (Molecular Devices), using a 20x/0.45 NA air objective lens and scientific CMOS camera (16-bit, 1.97mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTracker Green, Alexa fluor 555 and CellTracker Red, respectively. Images had pixel sizes of 0.68 à 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaXpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTracker channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the âcytoâ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns.15 Masks were used to generate cell outlines for intensity quantification. Figures were assembled with Adobe Illustrator.",
"appendix": "Data availability\n\nZenodo: Antibody Characterization Report for Transmembrane protein 106B, https://doi.org/10.5281/zenodo.7459629. 16\n\nZenodo: Dataset for the Transmembrane protein 106B antibody screening study, https://doi.org/10.5281/zenodo.7587647. 17\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina, Wolfgang Reintsch and Kathleen Southern\n\nABIF consortium: Claire M. Brown and Joel Ryan\n\nAn earlier version of the report can be found on Zenodo (https://doi.org/10.5281/zenodo.7459629).\n\n\nReferences\n\nManini A, Ratti A, Brusati A, et al.: TMEM106B Acts as a Modifier of Cognitive and Motor Functions in Amyotrophic Lateral Sclerosis. Int. J. Mol. Sci. 2022; 23(16). PubMed Abstract | Publisher Full Text | Free Full Text\n\nGallagher MD, Suh E, Grossman M, et al.: TMEM106B is a genetic modifier of frontotemporal lobar degeneration with C9orf72 hexanucleotide repeat expansions. Acta Neuropathol. 2014; 127(3): 407â418. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHu T, Chen Y, Ou R, et al.: Association analysis of polymorphisms in VMAT2 and TMEM106B genes for Parkinson's disease, amyotrophic lateral sclerosis and multiple system atrophy. J. Neurol. Sci. 2017; 377: 65â71. PubMed Abstract | Publisher Full Text\n\nVass R, Ashbridge E, Geser F, et al.: Risk genotypes at TMEM106B are associated with cognitive impairment in amyotrophic lateral sclerosis. Acta Neuropathol. 2011; 121(3): 373â380. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Blitterswijk M , Mullen B, Wojtas A, et al.: Genetic modifiers in carriers of repeat expansions in the C9ORF72 gene. Mol. Neurodegener. 2014; 9: 38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiang YX, Cao Q, Sawaya MR, et al.: Amyloid fibrils in FTLD-TDP are composed of TMEM106B and not TDP-43. Nature. 2022; 605(7909): 304â309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChang A, Xiang X, Wang J, et al.: Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases. Cell. 2022; 185(8): 1346â55.e15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nAlshafie W, McPherson P, Laflamme C: Antibody screening by Immunofluorescence.2021.\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358âD367. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25â38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAyoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021.\n\nAyoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021.\n\nStringer C, Wang T, Michaelos M, et al.: Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021; 18(1): 100â106. PubMed Abstract | Publisher Full Text\n\nAyoubi R, Fotouhi M, Ryan J, et al.: Antibody Characterization Report for Transmembrane protein 106B.2022. Publisher Full Text\n\nLaflamme C: Dataset for the Transmembrane protein 106B antibody screening study. [Data set]. Zenodo. 2023. Publisher Full Text"
}
|
[
{
"id": "173851",
"date": "02 Jun 2023",
"name": "Dawn Smallwood",
"expertise": [
"Reviewer Expertise Molecular biology",
"cell culture",
"medical genetics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful paper, highlighting the need for antibody validation. There is important information here for TMEM106B antibody users and clear methodology for those wishing to test antibodies in their own area. Experiments are well carried out and well controlled, and figures are clear. The lack of results analysis means that figures can be interpreted by the reader for their own experimental needs, however, any misinterpretation by a reader could be avoided by adding a column to Table 2 showing the study findings alongside âVendors recommended applicationsâ.\nThe number of technical replicates for each method should be stated.\nFull blots could be shown for immunoprecipitation results (Figure 2).\nI would have found figures easier to understand with consistent naming of HAP1 WT and KO cells as HAP1 WT and HAP1 KO rather than a combination of WT and THEM106B KO (Figure 1), HAP1 (Figure 2) and WT and KO (Figure 3).\nUnits could be added to the markers in Figures 1 and 2.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
},
{
"id": "183540",
"date": "04 Aug 2023",
"name": "Christian Peter Moritz",
"expertise": [
"Reviewer Expertise Western blotting",
"antibodies",
"autoantibodies",
"ELISA",
"method development"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn their article, Ayoubi and colleagues are reporting about their quality tests of six different commercial anti-TMEM106B antibodies using Western blotting, immunoprecipitation, and immunocytochemistry.\n\nThe article is interesting and useful for groups working with this protein.\nDoing KO controls to test antibodies is considered the gold standard of validation tests. Nevertheless, unfortunately it is not done very often. Thatâs why I appreciate the work of these authors.\nI would consider the following concerns.\nMAJOR CONCERNS:\n1) Chapter: âResults and discussionâ:\nIn my experience it is common to describe the results in the result chapter instead of just referring to the figures.\n\nThere is no discussion in this chapter yet. The authors should interpret their results in a more concrete way. Which antibodies do they recommend for which method?\nInteresting to mention in my eyes: 2 out of 6 tested antibodies donât work in Western blotting, as they donât bind the protein of interest, but they nonspecifically bind other proteins. One further antibody (60333-1-lg) nonspecifically binds other proteins in addition to binding the protein of interest.\nIn total, half of the antibodies show unspecific binding, which could harm result interpretation when using this antibody without having a KO control.\n\nArticle type \"Data Note\": In my perspective this article is not a \"data note\" in the classical sense (Journal info: \"Data Notes are brief descriptions of datasets that promote the potential reuse of research data and include details of why and how the data were created; they do not include any analyses or conclusions\").\nThe authors do not present a classical data set however. To me, an analysis and conclusions are necessary. Hence, I would ask the authors to consider another article type. Is there something like a \"Technical note\"?\nMINOR CONCERNS:\nIn their introduction, the authors speak of âa 135 amino acid portion of the protein from its luminal C-terminal domainâ, that might play a pathophysiological role.\nGiven this information, it might be interesting which of the six antibodies have epitopes in that specific domain. I there any information available? Some ideas to address this questions methodologically ?\n\nResults and discussion: âFor Western blot experiments, we resolved proteins from WT and TMEM106B KO cell extracts and probed them side-by side with all antibodies in parallel (Figure 1).â\nWhat means âside-by sideâ here? Do they come from the same membrane in order to avoid membrane-to-membrane variation? In the best case, in future experiments, the authors could even considering cutting the lanes vertically in order to even avoid lane-to-lane variation (cf. Pubmed ID 30768763).\n\nFigure 1, panel PA5-34353:\nI am a bit surprised that the âscratchesâ and inhomogenities in protein transfer is not equally visible in the total protein staining (ponceau). I would kindly ask the authors to check in their data files that the ponceau staining is really originating from the same membrane, because the loading control would be invalid otherwise.\n\nFigure 2: Why is âHCâ (antibody heavy chain) only shown in the second panel? I would recommend showing it in the first panel only, or in all panels.\n\nFigure 2: I do not understand why there is written âWB: 93334**â below each of the 6 Western blots.\n\nFigure 3: Is the Alexa-fluor 555 not interfering with the fluorescence stainings that were applied to distinguish WT from KO?\n\nConcluding sentence in the results section: âwe have screened many TMEM106B commercial antibodiesâ\nI recommend avoiding vague words like âmanyâ where a more concrete indication (âsixâ) is possible. Same for the word âseveralâ in the same sentence.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
},
{
"id": "178524",
"date": "04 Aug 2023",
"name": "Giuseppe Legname",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the various antibodies available for the study of TMEM106B. This is a useful methodological paper. The work is well organized, and the methodologies appropriately presented. I would like to suggest comparing the methods and results with those suggested by the vendors and discuss advantages to use the methodologies presented by the authors.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-308
|
https://f1000research.com/articles/12-307/v1
|
21 Mar 23
|
{
"type": "Case Report",
"title": "Case Report: Neglected case of duodenal strongyloidiasis in an immunocompromised patient",
"authors": [
"Smritee Mahat",
"Usha Manandhar",
"Pratima Thapa",
"Nabin Rokaya",
"Mohammad Rizwan Alam",
"Usha Manandhar",
"Pratima Thapa",
"Nabin Rokaya",
"Mohammad Rizwan Alam"
],
"abstract": "Strongyloides stercoralis, one of the common opportunistic parasitic infections, affects a large population in many parts of tropical and subtropical regions and is often a neglected tropical disease. The parasite completes its life cycle inside the human host with a unique feature of autoinfection, persisting in the host indefinitely in dormant form and having the notorious ability to cause fetal complications in an immunocompromised individual. Many factors like corticosteroid therapy, immunosuppressive therapy for autoimmune diseases, human T lymphotropic virus, malignancy, malnutrition etc. can transform asymptomatic strongyloidiasis to fatal outcomes, from hyperinfection syndrome to parasite dissemination leading to increased risk of high mortality rates. It has been neglected in screening unless and until some clinical suspicion appeared during the course of treatment. Early diagnosis and treatment can mitigate the rapid disease activation and evolution in an immunocompromised patient. A mandatory but reliable parasite screening method should always be carried out before administering immunosuppressants, even though simple stool tests and serology are only used when suspicion arises. Our case involved a 50-year-old female on cyclophosphane for systemic lupus erythematous (SLE) who experienced repeated bouts of vomiting and loose stools. Even several days after admission, there was no satisfactory outcome with medical management; a duodenal biopsy was carried out, which revealed numerous larvae of Strongyloides stercoralis.",
"keywords": [
"Cyclophosphane",
"Disseminated strongyloidiasis",
"hyperinfection",
"Immunosuppression",
"Strongyloidiasis",
"SLE",
"Strongyloides stercoralis",
"colitis."
],
"content": "Abbreviations\n\nGPIA: Gelatin particle indirect agglutination\n\nIFA: Indirect fluorescent antibody\n\nIHA: Indirect haemagglutination\n\nLIPS: Luciferase immuno-precipitation system\n\nPAP: Papanicolaou\n\nPCR: Polymerase chain reaction\n\nSLE: Systemic lupus erythematosus\n\nSsIR: Strongyloides stercoralis immuno-reactive antigen\n\nUGIE: Upper gastrointestinal tract endoscopy\n\nUSG: Ultrasonogram\n\n\nIntroduction\n\nStrongyloidiasis is a common parasitic opportunistic infection affecting millions of people worldwide, especially in tropical and sub-tropical zones. It has a unique ability to complete autoinfection and remains in dormant larval form for many years. Some of the factors leading to the immunocompromise state like corticosteroid therapy, human T-cell lymphotropic virus type-1 infection (HTLV-1), hematological malignancy, malnutrition, human immunodeficiency virus (HIV), among others, can activate and transform the dormant larval form into to widespread disseminated form with the end result of mortality in an unexpected manner. The practice of early screening for such parasitic infection in an immunocompromised patient has been neglected for many years unless and until some suspicious clinical presentation has drawn attention to the clinician. Therefore, in view of the above, the study of our case report draws the attention of many clinicians to the fact that early diagnosis of strongyloidiasis is one of the essential clinical practices in order to prevent the high risk of mortality due to negligence of parasitic screening in an immunocompromise patient.\n\nDuodenal strongyloidiasis is caused by a parasite, Strongyloides stercoralis, a soil-transmissible nematode. Strongyloidiasis is an infection caused by Strongyloides stercoralis (and rarely Strongyloides fuelleborni), which is a helminth present mainly in tropical and subtropical regions, as well as in temperate climates.1 The three different definitive hosts for Strongyloides stercoralis are humans, dogs, and cats. Around 3-100 million people are estimated to be infected worldwide.2 Strongyloidiasis has varied manifestations ranging from asymptomatic to potentially fatal hyper infection or disseminated infection. The female worm produces eggs by parthenogenesis that hatch to larvae, which may also cause auto-infection as well.3\n\n\nCase report\n\nA 50-year-old female, a known case of systemic lupus erythematous (SLE) under cyclophosphane, presented to emergency services with repeated bouts of watery vomiting for one week. She had repeated loose stools for one month. She was admitted for severe dehydration and dyselectrolytemia, for which she had been managed with an intravenous fluid like ringer lactate and antibiotics (metronidazole and ceftriaxone) for seven days. She was a housewife involved in agriculture occupation in the past. There was no family history of similar illnesses and other genetic diseases so far. Stool examination revealed cysts of Entamoeba histolytica. Ultrasonography (USG) of abdomen showed features suggestive of colitis and mild hydroureteronephrosis. Upper gastrointestinal endoscopy (UGIE) showed antral erosions, pangastritis, loss of mucosal folds in D2, esophageal candidiasis, and urease-positive Helicobacter pylori-induced chronic fundal gastritis. Computed tomography (CT) of the abdomen showed ill-defined, diffuse irregular luminal narrowing of ascending colon with a diffuse thickened wall. Endoscopic duodenal biopsy from the second part of the duodenum was done with suspicion of celiac disease. Histopathological examination showed duodenal mucosa revealing numerous larval forms and eggs of Strongyloides stercoralis within crypts and glandular epithelium in a background of dense eosinophilic infiltrates in lamina propria with no evidence of celiac disease or malignancy, as shown in Figures 1 and 2. After the diagnosis and constant lack of improvement in the condition, the patient opted for changing treatment and went to another hospital. Resection anastomosis of the intestine was done at another institute. Unfortunately, the patient died without any signs of cure.\n\n\nDiscussion\n\nStrongyloidiasis is a much-neglected health problem.4 Until the diagnosis of this ailment, most of the time, it has already been disseminated in the body. The causative organism for strongyloidiasis, Strongyloides stercoralis, is a helminth that gets transmitted by transdermal migration of filariform larvae, which is the infective form with hematogenous dissemination to the lungs. Through the tracheobronchial tree, it then enters the alveolar sacs, and the larvae gain access to the gastrointestinal tract.5 In the duodenum and jejunum, it hatches into rhabditiform larvae, which is the pathogenic form. Rhabditiform larvae, when passed in feces, can develop directly into free-living adult males and females that can copulate and release eggs or can instead molt two times to become infective filariform larvae and penetrate the skin to start a new parasitic cycle.3,6\n\nPatients are usually asymptomatic; however, gastrointestinal manifestations may mimic coeliac disease. The common symptoms are dyspepsia, abdominal discomfort, diarrhea, nausea, and small bowel obstruction. Intestines, particularly the duodenum and jejunum, are commonly affected sites that can present as small bowel obstruction.7 In patients with disseminated strongyloidiasis, pulmonary manifestations are also common, with the lung being the most common extraintestinal site to be involved. Pulmonary symptoms like dry cough, progressive dyspnoea, and wheezing are common in patients with pulmonary strongyloidiasis.5 Skin rash and pruritus are the dermal manifestations, and systemic symptoms of weight loss may also be noted in most cases.4 Immunocompromised conditions, HTLV-1, HIV infection, as well as patients under corticosteroids, present with more severe forms of infection.6 Some studies even indicate that corticosteroids accelerate the transformation of rhabdtitiform larvae to filariform larvae.1 The helminthic manifestation, though most common in the small intestine, can also occur at any level from the esophagus and stomach to the rectum.8 Duodenal aspirate or biopsy, though more sensitive for the diagnosis, is an invasive procedure, is less desirable.8,9\n\nS. stercoralis infection is difficult to differentiate from the infection caused by Ancylostoma duodenale, further requiring serological tests, stool examination, and culture.3,10 Adult worms or larvae are seen on stool examination. Detection of larvae in a smear of stool in saline is a definitive diagnostic feature; however, Papanicolaou (PAP) smears of sputum may also reveal the organism.5 Both S. stercoralis and A. duodenale are transmitted via soil.6 Eggs of S. stercoralis are oval, thin-shelled, resembling that of A. duodenale but are rarely seen in the stool. The non-infective form known as rhabditiform larva is the pathogenic form, which is the feeding stage with a length of 0.2-0.25 mm. They have a prominent genital primordium, a short buccal cavity, a clear esophagus, and a notched buccal opening.\n\nIn contrast, A. duodenale has an inconspicuous genital primordium and a long buccal canal. Filariform larva is the infective stage in both parasites, with a similar length of approximately 600 ÎŒ. However, S. stercoralis has a notched tail, and A. duodenale has a pointed tail. The esophagus to intestine ratio is 1:1 in S. stercoralis and 1:3 in A. duodenale.9,11 Enzyme-linked immunosorbent assays (ELISA), along with stool studies, are done together to increase the sensitivity of the investigation.6 Various immunodiagnostic methods like indirect haemagglutination (IHA), indirect fluorescent antibody (IFA), Western blot, ELISA, luciferase immuno-precipitation system (LIPS), gelatin particle indirect agglutination (GPIA), S. stercoralis immuno-reactive antigen (SsIR), and molecular methods like PCR are available.9,12 Culture methods for the proper identification of S. stercoralis, like Baermann and Koga agar plate techniques, are also available. The Kato Katz technique is employed for demonstration of A. duodenale.10\n\n\nConclusions\n\nStrongyloidiasis is one of the common parasitic infestations and is neglected in many developing countries. The infection is more prevalent among people with an immunocompromised state and clinically presents as nonspecific symptoms ranging from nonspecific gastrointestinal symptoms like abdominal pain, diarrhea, to fatal disseminated infection, thereby increasing the risk of unexpected mortality. Hence, while reviewing the cases, scrutiny is required not to miss out on the parasites. Also, before commencing the treatment with corticosteroids and other immunosuppressive drugs, the possibility of already existing parasites should be kept in mind so as to prevent the disease's activation, evolution, and progression to life-threatening or fatal complications. Hence, there is much need to transform the early screening of neglected common parasites into good clinical practice in immunocompromised patients in tropical and sub-tropical zones across the globe.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nOral consent was obtained from the patientâs family for the publication of the patientâs details.",
"appendix": "References\n\nKeiser PB, Nutman TB: Strongyloides stercoralis in the immunocompromised population. Clin. Microbiol. Rev. 2004 Jan; 17(1): 208â217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchÀr F, Trostdorf U, Giardina F, et al.: Strongyloides stercoralis: global distribution and risk factors. PLoS Negl. Trop. Dis. 2013 Jul 11; 7(7): e2288. PubMed Abstract | Publisher Full Text\n\nFraser J: A case report suggestive of strongyloidiasis infection occurring in temperate Australia. Rural Remote Health. 2019 May 1; 19(2): 161â164. PubMed Abstract | Publisher Full Text\n\nBuonfrate D, Requena-Mendez A, Angheben A, et al.: Severe strongyloidiasis: a systematic review of case reports. BMC Infect. Dis. 2013 Dec; 13(1): 1. PubMed Abstract | Publisher Full Text\n\nMokhlesi B, Shulzhenko O, Garimella PS, et al.: Pulmonary strongyloidiasis: the varied clinical presentations. Clin. Pulm. Med. 2004 Jan; 11(1): 6â13. PubMed Abstract | Publisher Full Text\n\nRamanathan R, Nutman TB: Strongyloides stercoralis infection in the immunocompromised host. Curr. Infect. Dis. Rep. 2008 Mar; 10(2): 105â110. PubMed Abstract | Publisher Full Text\n\nPatra AA, Nath P, Pati GK, et al.: Strongyloides infection presenting as proximal small intestinal obstruction. ACG Case Rep. J. 2019 Jun; 6(6): e00124. PubMed Abstract | Publisher Full Text\n\nKishimoto K, Hokama A, Hirata T, et al.: Endoscopic and histopathological study on the duodenum of Strongyloides stercoralis hyperinfection. World J. Gastroenterol: WJG. 2008 Mar 3; 14(11): 1768â1773. PubMed Abstract | Publisher Full Text\n\nPuthiyakunnon S, Boddu S, Li Y, et al.: Strongyloidiasisâan insight into its global prevalence and management. PLoS Negl. Trop. Dis. 2014 Aug 14; 8(8): e3018. PubMed Abstract | Publisher Full Text\n\nBisoffi Z, Buonfrate D, Montresor A, et al.: Strongyloides stercoralis: a plea for action. PLoS Negl. Trop. Dis. 2013 May 9; 7(5): e2214. PubMed Abstract | Publisher Full Text\n\nPage W, Judd JA, Bradbury RS: The unique life cycle of Strongyloides stercoralis and implications for public health action. Trop. Med. Infect. Dis. 2018 May 25; 3(2): 53. PubMed Abstract | Publisher Full Text\n\nCruz RJ, Vincenzi R, Ketzer BM: Duodenal obstruction-an unusual presentation of Strongyloides stercoralis enteritis: a case report. World J. Emerg. Surg. 2010 Dec; 5(1): 1â6. Publisher Full Text"
}
|
[
{
"id": "208615",
"date": "27 Sep 2023",
"name": "Catherine A. Gordon",
"expertise": [
"Reviewer Expertise Parasitology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction\nFirst paragraph\nReference first line â the most recent estimate is 600 million; Buonfrate, D. et al. The global prevalence of Strongyloides stercoralis infection. Pathogens 9, doi:10.3390/pathogens9060468 (2020).\nAlso delete opportunisitic.\nReview the lifecycle â Iâve put the CDC link below. It doesnât have a dormant form. It can be in a chronic stage, but the life cycle is still continuing, just a low output. The immunosuppression can then lead to hyperinfection due to immune dysregulation. So it is not dormant larvae that are transforming, it is the immune system not keeping control of the parasite burden and you get huge larval burdens. Hyperinfection is often accompanied by disseminated disease. https://www.cdc.gov/dpdx/strongyloidiasis/index.html#:~:text=Strongyloides%20stercoralis%20free%2Dliving%20adults.&text=Parasitic%20males%20do%20not%20exist%3B%20parasitic%20females%20are%20long%2C%20slender,adult%20worms%20(indirect%20cycle).\nActually, HIV doesnât seem to be particularly associated with the shift to hyperinfection, which is interesting.\nScreening would very much depend on a country or state basis. Thus, refer specifically to the country or area that you are referring to. Are their guidelines there for screening and are they just not being followed if yes?\nSecond paragraph\nYou mention twice in two sentences that strongyloidiasis is caused by Strongyloides stercoralis. Infection can also be due to Strongyloides fuelleborni kellyi as well. I would just say primarily by S. stercoralis. No need state Strongyloides stercoralis in full after the first mention, use S. stercoralis.\nActually, it is unknown if S. stercoralis is zoonotic. Dogs and cats can be infected with S. stercoralis, however, it is unclear if these are zoonotic. Some genetic testing has shown that there are different strains and that there are some strains found infecting dogs that also infect humans. However, dogs also practice coprophagy and thus it is unknown if this is from an active, patent infection, or from eating infected human faeces and the larvae making it through the dog in that way. So I would definitely not state with any confidence that animals are a definitive host.\nCheck ref above, more recent estimate is 600 million. Also, repetitious from first paragraph. As is the second sentence which states is present mainly in tropical and sub-tropical zones.\nLast sentence of paragraph, âwhich may also cause auto-infection as wellâ, the larvae are the ones causing autoinfection. Because they hatch inside the gut they can also develop into the infectious filariform larvae and penetrate the gut or skin around the anus to re-infect the host. So it is not as well.\nCase report\nFirst paragraph\nIs it watery vomiting or watery diarrhea?\nDelete âso farâ.\nEntamoeba histolytica is identical to the commensal organism Entamoeba dispar, you canât distinguish based on morphology, so was this further examined to speciate?\nIt is impossible to determine if these are Strongyloides larvae from these histology pictures. What else was done to determine if this was Strongyloides and not another gastrointestinal parasite? Was blood taken? Did she have eosinophilia? Was she tested either stool or blood test for Strongyloides? If she had diarrhea, was this examined for parasites?\nDiscussion\nDiagnosis can be made at any point of the disease not just when it is disseminated. There are serology tests that will detect in chronic phase, and faecal culture that will detect in acute and hyperinfection.\nThe larvae undergo tracheal migration, are coughed up and swallowed, thereby making it to the gut.\nThis whole first paragraph is just confused, and not written clearly or logically at all. If I was someone who knew nothing about Strongyloides I would read this paragraph and be incredibly confused and still not know anything about Strongyloides.\nNot sure if lungs are actually the most common site, it is part of their lifecycle so it makes sense that they are present in large numbers there in hyperinfection.\nHIV â does not seem to have the same affect as HTLV-I in the switch to hyperinfection.\nIs duodenal biopsy more sensitive for diagnosis? More sensitive than what?\nDid you do any differentiation in this case to determine if it was Strongyloides or Hookworm?\nNone of this about diagnostics is relevant or true.\nWhy are you talking about the morphology of Strongyloides so much? Did any morphological diagnosis occur in this case?\nAnd now we are talking about hookworm morphology, why?\nWhat use is listing all the potential diagnostics that were not used here?\nThe discussion has no actual discussion of the case.\nConclusion\nIt is not more prevalent in people who are immunocompromised. Being immunocompromised is a risk factor for developing more severe disease. All fine to say consider parasites, but was this patient actually treated for the parasite you think she had? There is no information on any treatment for any of the conditions she had.\nThis paper may be of interest and use in terms of screening. Because you canât just screen everyone if there is no indication. Not everyone who lives in the tropics or sub-tropics is at risk of Strongyloides infection, so there has to be criteria for screening. Carnino, L. et al. A practical approach to screening for Strongyloides stercoralis. Trop Med Infect Dis 6, doi:10.3390/tropicalmed6040203 (2021).\nWhat in the rest of this paper would support that need for early screening? I support it of course, but there is nothing in this paper that leads to that conclusion.\nI have now reached the end of the paper and I still donât know what country this case has occurred in. I can assume it is in Nepal based on the author affiliations. But what was the background of the patient? What was the likely method of infection, what risk factors did she have, what other test results did she have that may have indicated Strongyloides? If she was transferred to another hospital, how can you write the case report without the results of any testing or treatment they did at the other hospital? Assuming she was diagnosed with Strongyloides from the histology slides, was she treated for Strongyloides?\nTo add to all of this, English also needs some work.\n\nIs the background of the caseâs history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
},
{
"id": "259928",
"date": "04 Apr 2024",
"name": "Chamarika Jayanetti Weerasekera",
"expertise": [
"Reviewer Expertise Medical Parasitology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle Omit the word 'duodenal' - Strongyloides stercoralis is a small intestinal parasite and is found in the duodenum.\nMention the country.\nAbstract The abstract should stand alone independently but is not demonstrated in this case. Write about the salient features of the case report, briefly describing the importance.\nKeywords Cyclophosphane - Is it another term for the alkylating agent cyclophosophamide? If so, use the latter name that many in the medical community are familiar with\nIntroduction\nFirst paragraph How common is it- Buonfrate et al.,2020 estimated a prevalence more then 600 million in 2017.You can also mention the estimated prevalence for the Nepali general population in this case.\nThere is no dormant form in S.stercoralis (CDC - DPDx - Strongyloidiasis)\nAn association with HIV is controversial, although several research support it. See below\n[2],[3]\nThe last sentence of the first paragraph seems out of place. The way it is worded is more suitable for the discussion.\nSecond paragraph There is a possibility of a zoonotic reservoir, but currently, it is not definite. It even may vary with country to country. Mention the reference for the zoonotic reservoir.\nPlease study the life cycle well, prior to writing the introduction.\nIn general\nPlease rearrange the sentences. The last sentence of the introduction should lead to the opening of the case report.\nCase report\nPlease don't refer a patient as a \"case\".\nThe history and examination is inadequate. Please try to answer the following questions when revising the manuscript.\nIs it watery vomiting or diarrhoea? How long was she on treatment for SLE? How was the progression of the gastrointestinal symptoms. What was the systemic history? - especially symptoms related to the respiratory system? Was there any larva currens? Specifiy the dyselectrolytemia,of this patient What were the risk factors- specify Use the term faecal examination. You cannot specify species as Entamoeba histolytica cysts on morphology alone.Be cautious when using this terminology CDC - DPDx - Amebiasis)\n\nPlease answer the following questions when revising the manuscript What were the faecal examination techniques performed? Were the cultures done, if not, why? (the cultures are easy to perform and are done in Parasitology laboratories) Were molecular tests performed? What were the other tests done prior to endoscopy? Was the patient eosinophilic? Was the patient treated with any anthelminthics? If not, why? It is very important to mention what led to the patient's death.\nWas the colitis diagnosed only by ultrasonography? What was the relevance of the colitis, the presence of Entamoeba cysts and the clinical outcome of this patient?\n\nIt is always a good practice to use a timeline for clarity, especially in a complicated case like this.\nFigures Use arrows in the figures to point the features of interest.\nWhat was the basis for the identification of S.stercoralis by histology? Why cannot it be another parasite?\nDiscussion\nFirst paragraph Strongyloidiasis does not have to be disseminated prior to diagnosis. Diagnosis can be through cultures in acute or even in asymptomatic infection. Serology can also be used.\nThe larvae enter the lungs via the blood stream, then molt and ascend in the tracheobronchial tree, get swallowed in the pharynx and enter the gastrointestinal system.\nRhabditiform larvae are not mentioned as the only pathogenic form.\nWrite also about the autoinfective pathway. These are essential . However, if mentioned in the introduction, does not need to repeat in the discussion.\nSecond paragraph\nHow common is small bowel obstruction in strongyloidiasis?\nThere are two types of severe strongyloidiasis - hyperinfection and dissemination . In hyperinfection, symptoms are confined to the GI tract and respiratory system.\nWhy do you say duodenal aspirates are more sensitive? What is it compared against? Then what about the diagnostic tests done on the faecal samples and serology?\nThird paragraph\nS.stercoralis needs to be differentiated from hookworm - this includes both A.duodenale and Necator americanus. Direct microscopy is specific but less sensitive.\nEven a direct wet mount of sputum or bronchoalveolar fluid in hyperinfection can demonstrate the organism.\nPlease read this thoroughly CDC - Strongyloides - Resources for Health Professionals\nExtensive description of morphology is unnecessary.\nFourth paragraph\nExtensive description of the morphology is out of place\nSerology, although sensitive, is more likely to cause false positives due to cross reactions with other helminths in endemic settings.\nWhy is there a lot of emphasis on different serological techniques in this case?\nWhy is it needed to talk about Kato-Katz?\nIn general\nThe discussion needs to be relevant to the case report\nConclusions\nThis should highlight the importance of high clinical suspicion of strongyloidiasis in immunosuppressed patients with diarrhoea, increased awareness among doctors, proper diagnosis, with faecal examination and molecular techniques and prompt treatment.\nConsent Mention as informed consent\nOverall The English needs to be improved. The manuscript should be in an order. Please write in accordance to the CARE guidelines for case reports (CARE Checklist â CARE Case Report Guidelines (care-statement.org)) Given below are several case reports on strongyloidiasis written by some Sri Lankan authors. [5],[4]\n\nIs the background of the caseâs history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
},
{
"id": "259922",
"date": "02 May 2024",
"name": "Gilles Eperon",
"expertise": [
"Reviewer Expertise Tropical medicine"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nImportant case to report considering the rise of new immunosuppressive treatment while their safety profile are not studied in Stronglyoides endemic region⊠also it raises the need for screening before introduction of immunosuppressive treatment. But all the text need major revisions.\nMajor comments:\n\nAbstract should resume the article in structure and content. Moreover, the aim of publishing this case presentation is not formally written.\n\nCase description (clear exposition history : which country?, clear clinical presentation, history of immunosuppressive treatment, dosage of the medication, then why no antiparasitic treatment was not administrated, why sugrical procedure performed) should be more detailed.\n\nThe aim of the authors seems to raise the awareness of this Neglected Tropical Disease but most of the discussion is about morphological description what seems not to be the place for it. Moreover, a clear discussion about the type of diagnostic test and its value is not properly discussed. All possible way of detection is written without assessing their validity and place according to international recommendation. We do understand diagnostic tests are missing in most place, and probably in authorsâ place. It should be clearly stated and discussed, as it is one the main cause for the negligence of this disease. Moreover, most of disseminated strongyloidiasis is a failure of the screening test, as a proper screening test can confirm the diagnosis before the dissemination.\n\nThe life cycle is incorrect (switching term between rhabditiform & filariform?) and not well written. Please, revise it deeply.\nMinor comments:\nSome mistyping (ie âfetal complicationsâ in the astract) please read it again by an English-speaker and correct as needed\n\nDetails comments: Introduction Page 3: there is no dormant form; & consider update with recent estimate of the prevalence\nCase report Page 3: â⊠examination revealed cysts of Entemoeba histolytica.â Correct to âEntamoeba histolytica/disparâ as both species are microscopically similar Page 4, figure 2: please add arrows\n\nIs the background of the caseâs history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
},
{
"id": "281766",
"date": "05 Jun 2024",
"name": "Joelma Nascimento de Souza",
"expertise": [
"Reviewer Expertise Parasitic infections immunodiagnosis",
"specially S. stercoralis."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nABSTRACT The text needs a grammatical review. Furthermore, authors must add the manuscript aim, as well as a conclusion. âStrongyloides stercoralis, one of the common opportunistic parasitic infectionsâ - Since the 1980s, S. stercoralis is no longer considered an opportunistic parasite. This information is wrong. âis often a neglected tropical diseaseâ - Strongyloidiasis, according to the WHO, is a neglected disease. âThe parasite completes its life cycle inside the human hostâ - S. stercoralis has a double life cycle, one in the human host and other in the environment. âpersisting in the host indefinitely in dormant formâ - S. stercoralis does not enter a period of dormancy, it reproduces at low levels, which allows the infection to remain asymptomatic for decades. âfetalâ â It is fatal\n\nINTRODUCTION\nThe introduction is poorly written, with repeated information and no flow of information. âStrongyloidiasis is a common parasitic opportunistic infectionâ â Please, see comment above. âremains in dormant larval form for many yearsâ - Please, see comment above. âSome of the factors leading to the immunocompromise state like corticosteroid therapy, human T-cell lymphotropic virus type-1 infection (HTLV-1), hematological malignancy, malnutrition, human immunodeficiency virus (HIV)â - Currently, HIV infection is no longer considered a risk factor for severe strongyloidiasis. Please review this. âcan activate and transform the dormant larval form into to widespread disseminated form with the end result of mortality in an unexpected mannerâ - In fact, immunocompromise leads to an imbalance in the parasite-host relationship, stimulating the autoinfection cycle, causing hyperinfection and dissemination. I recommend this reference: (Ref-1) âAround 3-100 million people are estimated to be infected worldwideâ - Recent work estimates a frequency above 600 million. Reference: (Ref -2) âhatch to larvae, which may also cause auto-infection as well.â - In fact, it often causes autoinfection. It is also important to highlight that the parasite is released in the feces in the form of larvae, which requires special methods for parasitological diagnosis, such as Baermann-Moraes and Agar Plate Culture.\nCASE REPORT âa known case of systemic lupus erythematousâ - How long ago was the diagnosis made? What was the clinical presentation? Was the patient symptomatic? âunder cyclophosphaneâ - What is the dosage? ârepeated bouts of watery vomiting for one week. She had repeated loose stools for one month.â Â - Did the patient only experience vomiting and diarrhea? Did she not have abdominal pain or fever? It is important to deny it. âStool examination revealed cysts of Entamoeba histolyticaâ - Which parasitological method was used? âcysts of Entamoeba histolyticaâ - cysts of Entamoeba histolytica âHelicobacter pylori-inducedâ - Helicobacter pylori-induced âAfter the diagnosis and constant lack of improvement in the conditionâ - What treatments were performed for S. stercoralis infection? For how long? Remembering that the patient also had E. histolytica cysts and H. Pylori that needed to be treated. Were these conditions also managed? âUnfortunately, the patient died without any signs of cure.â How long did it take for the patient to die after the onset of symptoms? How long after the last treatment? What is the official cause of death? Figure 2 - Please point to the Strongyloides eggs and larvae with arrows, making everything clear in the description as well as the morphological characteristics that lead to S. stercoralis identification.\nDISCUSSION The discussion needs extensive review. Several information is inaccurate. Furthermore, the author barely discusses the case report presented. âThrough the tracheobronchial tree, it then enters the alveolar sacs, and the larvae gain access to the gastrointestinal tract. In the duodenum and jejunum, it hatches into rhabditiform larvae, which is the pathogenic form.â - After the pulmonary cycle, the parasite establishes itself in the small intestine, where it reaches the adult stage: parthenogenetic females. The females will reproduce by parthenogenesis, releasing eggs, which transform into larvae while still inside the host. Rhabditoid larvae are not the pathogenic form. Please review this and rewrite the paragraph. âIn patients with disseminated strongyloidiasis, pulmonary manifestations are also common, with the lung being the most common extraintestinal site to be involved.â - The pulmonary tract is part of the autoinfection cycle, with pulmonary involvement being common in symptomatic individuals. Please review this. âDuodenal aspirate or biopsy, though more sensitive for the diagnosis, is an invasive procedure, is less desirable.â- There is no indication for duodenal aspirate or biopsy as diagnostic methods for S. stercoralis infection. Generally, the parasite is an incidental finding. Please review this. âS. stercoralis infection is difficult to differentiate from the infection caused by Ancylostoma duodenaleâ - It is difficult to differentiate not only from A. duodenale, but also from other species of the Ancylostomatidae family. Please, review this. âAdult worms or larvae are seen on stool examinationâ - Generally, S. stercoralis adult worms are not seen in feces. Please, review this. âDetection of larvae in a smear of stool in saline is a definitive diagnostic featureâ â Direct smear has a very low sensitivity. Please, review this. âThe non-infective form known as rhabditiform larva is the pathogenic formâ - I didn't understand what the author meant by pathogenic form. Please clarify this. âVarious immunodiagnostic methods like indirect haemagglutination (IHA), indirect fluorescent antibody (IFA), Western blot, ELISA, luciferase immuno-precipitation system (LIPS), gelatin particle indirect agglutination (GPIA), S. stercoralis immuno-reactive antigen (SsIR)â â SsIR is a recombinant antigen, not a immunodiagnostic method. Please, review this. âCulture methods for the proper identification of S. stercoralis, like Baermannâ â Baermann-Moraes is a parasitological test but not a culture method. Please, review this. âThe Kato Katz technique is employed for demonstration of A. duodenaleâ - Kato Katz has a very low sensitivity for hookwormsâ diagnosis. Please, review this.\nCONCLUSIONS It needs an overall revision as well as a conclusion to the case presented.\n\nIs the background of the caseâs history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-307
|
https://f1000research.com/articles/12-305/v1
|
20 Mar 23
|
{
"type": "Research Article",
"title": "A first Tunisian pilot study investigating sexual dysfunctions in patients with hemophilia.",
"authors": [
"Kmira Zahra",
"Emna Bouselama",
"wided cherif",
"rim aidli",
"Rania Bannour",
"badii amamou",
"neila fathallah",
"monia zaier",
"nesrine ben sayed",
"yosra ben youssef",
"Haifa Regaieg",
"Abderrahim Khelif",
"Emna Bouselama",
"wided cherif",
"rim aidli",
"Rania Bannour",
"badii amamou",
"neila fathallah",
"monia zaier",
"nesrine ben sayed",
"yosra ben youssef",
"Haifa Regaieg",
"Abderrahim Khelif"
],
"abstract": "Background: Little data is actually available on sexual health problems with sexual activity, and intimacy (sexual difficulty) in patients with hemophilia (PWH). We conducted this study to determine the prevalence of sexual difficulty in PWH and to determine factors associated with erectile dysfunction (ED). Methods: Based on The International Index of Erectile Function 15 (IIEF-15) questionnaire, we evaluated ED and other sexual problems in PWH. Results: Forty-Three (43) PWH were included in our study. The mean age was 33 years. Fourteen (32.6%) respondents were identified as having severe disease. The majority (93%) suffered from erectile dysfunction. Lack of desire and orgasm was observed in 76.7% of cases each. Lack of sexual satisfaction and global satisfaction were noted in 83.7% of cases and 88.4% of cases, respectively. Among PWH, older age was associated with ED. Conclusion: Our study illustrates the need for programs to assess and improve the sexual health of PWH in comprehensive hemophilia care.",
"keywords": [
"Hemophilia",
"Patient-reported outcome",
"Erectile dysfunction",
"Sexual health."
],
"content": "Background\n\nSexuality is a keystone of human beings and life quality. Sexual activity is complex and usually involves physical, psychological, emotional, and hormonal status.1,2 Limits to incorporating sexual health into comprehensive care for patients with hemophilia (PWH) include provider and patient comfort with sexual health and activity.3 Moreover, cultural and social factors may deeply influence the comfort level of the healthcare provider and the patient discussing sexual health.4 Thus, in PWH, knowledge about sexual health, difficulty with sexual activity and intimacy (sexual difficulty) is not enough explored and is extremely restricted.4,5 Based on our personal experiences with patients in one side, and on discussions with hemophilia providers in the other side, we supposed that sexual health is insufficiently and not routinely assessed or discussed at our center. This led us to conduct, to the best of our knowledge, this first Tunisian study to investigate the specific types of sexual issues experienced in PWH and to assess factors associated with erectile dysfunction (ED).\n\n\nMethods\n\nThis is an observational study performed at the regional hospital of Farhat Hached, Sousse, Tunisia. PWH registered at the local register met the following eligibility criteria, were included: aged 18 or over, and did not require special assistance to complete the questionnaire. Exclusion criteria involved PWH associated with other bleeding disorders and those who refused to answer the questionnaire. Eligible patients were invited to join the study through telephone contacts. Data were collected using the questionnaire of the International Index of Erectile Function IIEF15 in its Arabic version6,7 and by using a clinical record created to collect clinical and sociodemographic data, via interviews and medical records search.\n\nThe IIEF 15 contains 15 items addressing the relevant domains of male sexual function that is erectile function, orgasmic function, sexual desire, and sexual satisfaction. The questionnaire is composed of five parts7:\n\n- Erectile function (comprised of 6 questions Q1, 2, 3, 4, 5 and 15): the domain of erectile function allows this graduation of the ED:\n\nâ Normal: score 26-30.\n\nâ Mild: score 22-25.\n\nâ Mild to moderately severe: score 17-21.\n\nâ Moderately severe: score 11-16.\n\nâ Severe: score less than 10.\n\n- Orgasmic function (comprised of 2 questions Q9 and 10): the score was considered abnormal if <9.\n\n- Sexual desire, (comprised of 2 questions Q11 and 12): the score was considered abnormal if <9.\n\n- Sexual satisfaction (comprised of 3 questions Q6, 7 and 8): the score was considered abnormal if <13.\n\n- Global satisfaction (comprised of 2 questions Q13 and 14): the score was considered abnormal if <9.\n\nThe results obtained were analyzed by the SPSS (Version 26). For quantitative variables, normality was tested by the Chi squared test. The qualitative variables are expressed in counts and percentages and were tested by the t student test. The logistic regression model was conducted to establish the association between ED and variables as possible predictors. The significance level was established at <0.05.\n\nEthically, all the patients included gave their consent to answer the questionnaire after being informed of the objective of the study and the anonymity of the questionnaire. We preferred oral consent to written one because it was more practical and easily feasible. Moreover, as we are dealing with sexual issues, we thought that oral consent allowed us to safeguard the participantâs anonymity and to reassure participants for the anonymity of their personal information. In all cases, the research and the purposes of this research were fully described to participants. Verbal informed consent has been obtained from all participants in the study in the following way: All the participants have received written invitations, which described and specified the purpose and terms of the study. When the participants showed up on the scheduled day for the interviews and the dialogue workshop, the conditions were repeated verbally with a specific emphasis on the measures taken to ensure confidentiality and anonymity in the report/article. This study and the collection of the different interview data was approved to be in accordance with standards for good scientific practice by the Committee for Research Ethics at Farhat Hached University Hospital, Tunisia (Ref: CER: 29-2022).\n\n\nResults\n\nWe included in our study 50 eligible patients. Seven patients were excluded as they didnât complete the questionnaire. 43 patients were finally retained and were interviewed, from June to August of 2022, representing 86% of the total. The majority (88.4%) was suffering from hemophilia A and 11.6% were suffering from hemophilia B.\n\nIn our population study, mean age was 33 years. The most important age group was 30â39 years old (41.8%). Almost half of patients were single (51.2%), not having children in 41.9% of cases, and unemployed in 55.8% of cases. Fourteen (32.6%) respondents were identified as having a severe disease, twenty (46.5%) with moderate disease and nine (20.9%) with mild disease. All patients suffered from hemophilic arthropathy but at varying stages. The most affected joint was the knee. Co-morbidities included Human immunodeficiency virus (HIV) and Hepatitis type B and Diabetes Mellitus in one patient respectively.\n\nTable 1 illustrates socioeconomic, demographic and clinical characteristics of PWH included in our study.\n\nIn the IIEF 15 questionnaire, the general average score was 46. Table 2 shows the average scores of each domain of the IIEF-15 questionnaire. ED was observed in 93% of cases; it was severe in 20.9% of cases (Figure 1).\n\nLack of desire and orgasm was observed in 76.7% of cases each. Lack of Sexual satisfaction and global satisfaction were noted in 83.7% of cases and 88.4% of cases, respectively (Table 2).\n\nED was significantly associated with participants' age (p=0.034). In fact, older age was associated with ED. Neither the clinical factors nor the other sociodemographic factors (overweight, level of education, professional and marital status) were associated with ED and this is probably due to the reduced number of our patients (Tables 3 and 4).\n\n\nDiscussion\n\nTo the best of our knowledge, we herein report the first pilot study of various sexual difficulties using the IIEF score in PWH. IIEF 15 is a linguistically validated questionnaire that has been reproduced in l0 languages and is readily self-administered in research or clinical settings. It has a high sensitivity and specificity in treatment-related changes in patients with ED.6,7\n\nCurrently, data related to sexual health in PWH are lacking. Sexual difficulty in PWH is not sufficiently discussed in routine hemophilia care because of many factors including lack of awareness, understanding and resources.8 In one hand, physicians may be hesitant to enquire about patientâs sexual issues and in the other hand, patients are often embarrassed to discuss about their sexual problems.9 Lower levels of knowledge in the field of sexual activity among adultâs males with hemophilia were reported in a Canadian study.10 This lack of information is also reported in the pilot study performed by Tobase et al.11 In fact, thirty percent of respondents thought they did not have satisfactory information concerning sexual activity in light of their bleeding disorder and roughly two-thirds (63%) of respondentsâ desire to be informed about sexual health issues at their consultation. Based on these facts and according to our personal experiences with patients and discussions with hemophilia providers, we supposed that sexuality is also insufficiently assessed or discussed at our center. This led us to carry out this study to reveal if there is sexual health impairment in our PWH and therefore trying to improve their sexual health.\n\nA proper understanding of the expectable sexual complications of hemophilia, the related comorbidities, and sexually related psychological issues are the essential elements required for sexual healthcare for PWH.\n\nTobase et al., investigating sexual health by using a 54-item patient reported questionnaire,11 demonstrated that forty percent (8 out of 20) of respondents believed that their bleeding disorder had a negative impact on their sexual life. In fact, a considerable proportion of PWH bleeds related to sexual activity. However, in this study, sexual difficulty in PWH was not detailed.\n\nPsychosocial issues reported by patients suffering from moderate to severe hemophilia were reported in the analysis of The Hemophilia Experiences, Results and Opportunities (HERO), it was reported that a significant number of young adults with hemophilia (37%/32%) thought that this affection disturbed their ability to make close relationships with a partner or prospective partner.5 The sexual difficulties were not detailed in this analysis.\n\nBased on Germini F. et al. study, predictive factors and prevalence of sexual issues in PWH were assessed.4 In this report data was analyzed from the Patient Reported Outcomes Burdens and Experiences (PROBE), which is a 29-item questionnaire dealing with patientâs health status and quality of life.4,12 In this report, between January 2016 and February 2017, 3979 adults were enrolled from 48 countries. Sexual difficulties were found in a total of 302 PWH (15, 1%). PWH with bleeding events were 3, 82 times experiencing sexual problems.4\n\nIn quality-of-life studies in PWH, erectile dysfunction and generalized sexuality were reported.10,13 Erectile function is classified as organic, psychogenic, or mixed organic and psychogenic.14 In most cases of ED, an organic basis is generally found,15 typically vascular in nature,16 but psychological factors are also frequently present.4 It is crucial to differentiate organic ED from psychogenic one. Classic organic ED is associated with diminished erections in all situations. In contrast, men with classic psychogenic ED have evidence of intact erectile function in certain situations, such as with self-stimulation or occasionally upon awakening. In some cases, and in the absence of evidences of an organic or psychogenic pattern, further laboratory investigation is required. Our study demonstrated ED in 93% of cases, moderately severe, and severe in 16.30% and 20.9%, respectively. This result can be explained by the frequency of arthropathy in our patients and probably by the frequency of bleeding which unfortunately was not studied in our patients.\n\nSeveral studies have tried to determine contributing factors of sexual difficulties in PWH.4,17,18\n\nAcute and/or chronic pain has a negative impact on physical and emotional performance. Patients suffering from chronic pain have, for the most of them (73%), pain-related difficulty with sexual activity.17 Sexual functioning is also affected by the consequences of pain management.18\n\nMoreover, bleeding was a great dilemma. Data found that PWH have more frequently recurrent sexual difficulties in cases of bleeding events occurring prior two weeks or having limb-threatening bleeding in the previous year.8\n\nIn addition, joint pain, restriction and lack of mobility can restrict sexual intercourse and sexual position resulting in sexual difficulty.19,20\n\nComorbidities impacting sexual function were also evaluated in the study performed by Germini and al.4 Hemophilia patients with underlying diseases such as viral hepatitis, HIV, diabetes Mellitus, high blood pressure and arthritis were approximately two times more likely to have sexual difficulties. In our study, because of limited number of patients (comorbidities in only 1 patient, and a positive serology for infections with contagious diseases in only 2 patients) didnât allow us to study their correlation with different domains of IIEF 15.\n\nAdditionally, it was demonstrated that severe disease in PWH increases two times the risk of sexual problems.4\n\nMulticenter inclusion will allow investigating contributing factors of sexual difficulty.\n\nWe suppose that this pilot study provides main insights and highlights into a poorly understood aspect of our patientâs sexual health. Nonetheless, our data indicate that further studies are needed to evaluate sexual health knowledge and experience in a larger cohort of PWH, including a younger and more diverse population, to enable us to develop and provide appropriate comprehensive care for our patients.\n\n\nConclusion\n\nFortunately, PWH are living longer and healthier lives, which allows the focus of comprehensive care to address all the essential health requirements including physical, mental and sexual health. Barriers to incorporating sexual health into comprehensive care for PWH include provider and patient comfort with the topic of sexual health. Having knowledge about sexuality in PWH is important in order to inform clinicians, other healthcare providers, and stake holders involved with policy development and comprehensive hemophilia care. The ultimate goal is to improve sexual health and well-being in PWH.",
"appendix": "Data availability\n\nFigshare. The International Index of Erectile Function Questionnaire Arabic Version, DOI: 10.6084/m9.figshare.22154945.v1. 21\n\nFigshare. The International Index of Erectile Function Questionnaire English Version, DOI: 10.6084/m9.figshare.22155044.v1. 22\n\nThis project contains the following data:\n\n- The International Index of Erectile Function\n\n- Questionnaire (IIEF) contains 15 questions about sex difficulty.\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC BY 4.0 Public domain dedication).\n\n\nAcknowledgment\n\nThe authors would like to thank the participants in the study.\n\n\nReferences\n\nParish KL: Sexuality and haemophilia: Connections across the life-span. Haemophilia. 2002; 8: 353â359. PubMed Abstract | Publisher Full Text\n\nFugh-Berman A, Scialli AR: Testosterone and sexual function. Curr. Opin. Urol. 2017; 27: 516â518. Publisher Full Text\n\nReinicke K, SÞgaard IS, Mentzler S: Masculinity Challenges for Menwith Severe Hemophilia. Am. J. Mens Health. 2019; 13(4): 155798831987262â155798831987211. Publisher Full Text Reference Source\n\nGermini F, Chai-Adisaksopha C, Pete D, et al.: Evaluation of the sexual health in people living with hemophilia. Haemophilia. 2021; 27: 993â1001. PubMed Abstract | Publisher Full Text\n\nBlamey G, Buranahirun C, Buzzi A, et al.: Hemophilia and sexual health: results from the HERO and B-HERO-S studies. Patient Relat. Outcome Meas. 2019 Aug 14; 10: 243â255. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRosen RC, Riley A, Wagner G, et al.: The International Index of Erectile Function (IIEF): a multidimensional scale for assessment of erectile dysfunction. Urology. 1997; 49: 822â830. PubMed Abstract | Publisher Full Text\n\nNeijenhuijs KI, Holtmaat K, Aaronson NK, et al.: The International Index of Erectile Function (IIEF)âA SystematicReview of Measurement Properties. Psychometrics. 2019; 16: 1078â1091. Publisher Full Text\n\nGianotten WL, Heijnen L: Haemophilia, aging and sexuality. Haemophilia. 2009; 15: 55â62. PubMed Abstract | Publisher Full Text\n\nWylie K, Kenney G: Sexual dysfunction and the ageing male. Maturitas. 2010; 65: 23â27. Publisher Full Text\n\nArnold E, Lane S, Webert KE, et al.: What should men living with haemophilia need to know? The perspectives of Canadian men with haemophilia. Haemophilia. 2014; 20: 219â225. PubMed Abstract | Publisher Full Text\n\nTobase P, Mahajan A, Francis D, et al.: A gap in comprehensive care: Sexual health in men with haemophilia. Haemophilia. 2017; 23: e389âe391. PubMed Abstract | Publisher Full Text\n\nMw S, Chai-Adisaksopha C, Curtis R, et al.: The Patient Reported Outcomes, Burdens and Experiences (PROBE) Project: development and evaluation of a questionnaire assessing patient reported outcomes in people with haemophilia. Pilot Feasibility Stud. 2018; 4: 58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWitkop M, Guelcher C, Forsyth A, et al.: Treatment outcomes, quality of life, and impact of hemophilia on young adults (aged 18-30 years) with hemophilia. Am. J. Hematol. 2015; 90(Suppl 2): S3âS10. PubMed Abstract | Publisher Full Text\n\nLue TF: Erectile dysfunction. N. Engl. J. Med. 2000; 342: 1802â1813. Publisher Full Text\n\nSullivan ME, Keoghane SR, Miller MA: Vascular risk factors and erectile dysfunction. BJU Int. 2001; 87: 838â845. Publisher Full Text\n\nAversa A, Bruzziches R, Francomano D, et al.: Endothelial dysfunction and erectile dysfunction in the aging man. Int. J. Urol. 2010; 17: 38â47. Publisher Full Text\n\nAmbler N, De CWilliams AC, Hill P, et al.: Sexual difficulties of chronic pain patients. Clin. J. Pain. 2001; 17: 138â145. Publisher Full Text\n\nNusbaum MRH, Hamilton C, Lenahan P: Chronic illness and sexual functioning. Am. Fam. Physician. 2003; 67: 347â354. PubMed Abstract\n\nBar-Chama N, Snyder S, Aledort L: Sexual evaluation and treatment of ageing males with haemophilia. Haemophilia. 2011; 17: 875â883. PubMed Abstract | Publisher Full Text\n\nRosenbaum TY: Musculoskeletal pain and sexual function in women. J. Sex. Med. 2010; 7: 645â653. Publisher Full Text\n\nZahra K: IIEF-15 Arabe (1).docx. figshare. Figure. 2023. Publisher Full Text\n\nZahra K: ILEF QUESTIONNAIRE ENGLISH. figshare. Figure. 2023. Publisher Full Text"
}
|
[
{
"id": "206340",
"date": "27 Nov 2023",
"name": "Marijo VodanoviÄ",
"expertise": [
"Reviewer Expertise Coagulation",
"hemophilia",
"thrombosis",
"benign hematology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere is no data about hemophilic arthropathy, prophylaxis, inhibitors against factors, and previous hepatitis B and C infection.\nWhich prophylaxis is being used (plasma derived factors, recombinant or bypassing agents, bispecific antibodies emicizumab etc).\nPWH and arthropathy have higher possibilty to bleed into joints in spite of prophylaxis.\nUnivariant and multivariant statistical analysis could give us correlation between arthropathy, prophylaxis, hepatitis and ED among PWH.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "337508",
"date": "21 Nov 2024",
"name": "Mustafa Suat Bolat",
"expertise": [
"Reviewer Expertise Andrology",
"endourology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReviewer comments\nBackgrounds:\nStarting with a concise statement about the importance of sexual health in general and then narrowing to its relevance for PWH to provide a logical progression might enhance better understanding.\nThe second comment is too difficult to understand. Please improve it.\nThe authors state that cultural and social factors may deeply influence the comfort level of the healthcare provider and the patient discussing sexual health. Thus, in PWH, knowledge about sexual health, difficulty with sexual activity and intimacy (sexual difficulty) is not enough explored and is extremely restricted. I did not understand relationship between two comments.\nThe authors investigated sexual functions in patients with hemophilia. Why do PWH are in increased risk of sexual dysfunction?\nMethods\nInclusion criteria is very limited. There should be information regarding;\nOther comorbidities History of sexual life Hormonal disturbances Past major pelvic surgery or radiotherapy Active sexual life Marital status Psychiatric diseases Drug use Alcohol and smoking habits. If present, duration of sexual dysfunction.\nNo of patients is very low. Did the authors perform power analysis?\nWhat do you mean with âThe most important age group was 30â39 years old (41.8%).â?\nDiscussion Authors should compare their findings with those in the literature and discuss possible reasons for any differences.\nConclusion The first comment seems like a continuation of an earlier comment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-305
|
https://f1000research.com/articles/12-301/v1
|
20 Mar 23
|
{
"type": "Opinion Article",
"title": "The house as a mind",
"authors": [
"Clive Gamble"
],
"abstract": "Palaeoanthropologists and evolutionary psychologists have successfully used the increasing size of the brain during human evolution to infer cognitive and social outcomes. Archaeologists have applied similar reasoning to the development of technology in deep history. This paper goes beyond these approaches by considering the house as a metaphor for the structure of hominin minds. It is argued that the study of the mind in deep history requires, (1) a recognition that mind is distributed between bodies, brains, and the world. The implications are examined through a magnetic resonance imaging (MRI) study (that unwraps the cerebellum and which suggests that folding rather than cortex size may be more important for understanding cognition.; (2) unmasking the ingrained container-habitus that has been used to describe and investigate minds either in the present or deep past. This bias is explored by entering the eccentric house-mind of Sir John Soane (1753-1837) with its many compartments, paintings, and antiquities; and (3) an exploration of alternative embodied metaphors to enable archaeologists to study distributed mind in deep history. The metaphor ARCHITECTURE WITHOUT WALLS is discussed and briefly compared to the evidence for âhousesâ in the Middle and Upper Pleistocene. The evidence indicates that hominins have always had complex, distributed minds but only recently in our deep history did we come to think predominantly through and with artificial containers such as houses. Late in human history these constructions became a common-sense habitus that expressed and fashioned our cognitive experience of the world.",
"keywords": [
"Deep history",
"containers",
"Neocortex",
"cerebellum",
"houses",
"Palaeolithic",
"human evolution"
],
"content": "Introduction\n\nHouses have agency because, as Alfred Gell declared, the house is a body for the body (Gell, 1998: 252-3). Houses act as bodies because, like bodies, they are containers with surfaces, entrances, and exits. They have organs of sense and expression; gaudy skins, spyholes to peer through and voices which reverberate through the night. Gell concluded that, âTo enter a house is to enter a mind, a sensibilityâ (ibid: 253). The question is, what kind of mind?\n\nIn this paper I examine three metaphors of containment and their implications for cognition and mentalizing in deep history. These are the embodied metaphors of THE MINDâS EYE, THE WALLS HAVE EARS, and THE DISTRIBUTED MIND. I also examine the house as a practical metaphor of embodiment using a free-to-visit example, Sir John Soaneâs Museum in London. I have two aims. First, to unmask a container-habitus in the study of contemporary and historic humanity. Second, to investigate the distributed mind through a more appropriate practical metaphor, ARCHITECTURE WITHOUT WALLS, for a Pleistocene-scale deep history. With this perspective the house, as an object of archaeological enquiry, provides an opportunity to understand how cognition evolved, not simply as a function of larger brains but through the bodily experience of containment.\n\nHouses were once, like minds, private, locked places. Access was by invitation only and a physical visit was needed. No longer. The internet allows us via house selling sites [Rightmove (rightmove.co.uk/), On the Market (onthemarket.com/), and Zoopla (zoopla.com/)] to enter thousands of properties at the click of a mouse. Like field-archaeologists, we can explore their contents, measure their floorplans and infer the owners hopes, dreams, and economic status, even give them a psychological profile. If houses are, as Gell suggests, minds, then there has never in humanityâs history been such open access and so many minds to explore. And all without a person visible. We understand minds in this online estate through the agency of things and our cognitive belief that houses, like minds, contain them. And it is this container-habitus which shapes our world and our past.\n\nTo cross the threshold of Sir John Soaneâs Museum in Lincolnâs Inn Fields, London, is to enter the mind of an architect and avid collector of classical antiquities (soane.org/). Soane zealously curated every detail of his career in an attempt, according to Sophie Thomas (2018: 131), âto fortify, document, and defend his lifeâ. She correctly describes him as an archivist of the self. Soaneâs house-museum is a biographical bundle, frozen in time. Its warren of rooms and spaces creates a house within a house into which his stuff was poured, then sealed by his will. But if we only regard his museum as a time-capsule of a life lived between 1753 and 1837, we miss the point of how Soaneâs houses and their contents work as a mind.\n\nSoaneâs houses are containers which resemble the disembodied mind inherited from René Descartes (1596-1650) with its interior spaces and machine-like pipes and pumps regulated by an internal overseer; a social architecture revealed by bisecting the brain (Figure 1).\n\nThe image appeared in Descartesâs Treatise of Man published posthumously in 1662.\n\nAt the centre of this boxed-in-brain sits the pineal gland, Descartesâs seat of reason and âcommonâ sense, but now caricatured by Daniel Dennett (1991: 106-7) as neither the turnstile of consciousness nor the Oval Office of the brain. Nonetheless, its central, enclosed position within the brain produced a powerful metaphor to express this containment, THE MINDâS EYE (Table 1), which Descartes used in the Regulae, published between 1619 and 1628.\n\nThese rules still underpin the methods used by most scientists.\n\nThe pineal gland and THE MINDâS EYE point to Descartesâs unspoken reliance on containers to express his cognitive system metaphorically. As summarised by George Lakoff and Mark Johnson (1999: 395) this has handed down to us three embodied metaphors which still dominate the description of how minds are understood. These are, THE MIND IS A CONTAINER OF IDEAS, IDEAS ARE OBJECTS, and KNOWING IS SEEING. And these are not just semantic metaphors. Sir John Soaneâs Museum is also a practical container metaphor, equivalent to Chris Tilleyâs (1999) concrete metaphor, expressed in material form (Gamble, 2023 in press). It is an example of a house-mind that is a compartmentalized, practical metaphor containing ideas, objects, and knowledge.\n\nDescartes and most who followed him worked in what I refer to as a world of container-habitus; an unrecognized bias to think predominantly in terms of the bodily experience of containment, enclosure, and compartmentalization. Tim Ingold with customary perspicacity has unmasked this dominant cognitive trait,\n\nâThis experience of containment influences our thinking about what it means to inhabit a world to an extent that even psychologists and philosophers, who are tasked with the investigation of such matters, are ill prepared to recogniseâ (Ingold, 2015: 41).\n\nArchaeologists can be added to Ingoldâs list of those who use, but do not recognise the formative influence of containment on cognition. However, archaeologists are not usually expected to do humanityâs thinking about what it means to inhabit a world. That has been left to those disciplines like anthropology and sociology which can speak with their subjects, or to historians who have access to minds through texts and archives. Rather, our primary task has always been to understand the history of inhabiting through things.\n\nThis role needs to change. Archaeologists can access hominin minds through metaphors that are older than language which shapes cognition with its semantic metaphors. We do this through practical metaphors, stuff. But as I have shown elsewhere (Gamble, 2007) the stuff which contains humanity developed and increased during deep history. The Neolithic sees an exponential rise in containers among them villages of mud-brick houses, graves in cemeteries, pots, stone bowls, textiles, bags, baskets, and more people (Gamble, 2023 in press). An explosion that outstrips arguments that selective preservation of perishable materials has skewed the evidence. Today we inhabit a hyper-container world where we live, in James Gibsonâs words, âboxed-up livesâ(1979: 203). Archaeologists reflect this unconscious, culturally inherited container-habitus by tracing the origins of modernity to the Neolithic explosion of container artefacts. This is apparent in the prehistories of Gordon Childe (1942) and Colin Renfrew (1973, 1996) while the same container-habitus is found throughout Yuvral Harariâs (2014) global history. The container-habitus in these narratives may be camouflaged by economic and symbolic smokescreens, but a puff of wind reveals the origin of our âboxed-up livesâ to be in âboxed-up prehistoric livesâ.\n\nNone of this is surprising. If we lift the lid on the Neolithic box of deep history it smells of home. This is not the case with the Palaeolithic box. Not because of smaller brains and the presence of hyenas but because there is much less stuff-that-contains inside its box. It is for us a strange world where our familiar cognitive props, those practical metaphors of containment, elude us.\n\nHaving recognised the container-habitus what can be done about it? In Sir John Soaneâs house-mind-museum we shouldnât be deceived by the solid walls which partition the spaces room by room, floor by floor; from the sarcophagus of Seti Ist in the basement to his architectural drawings for the Bank of England in the attic (Figure 2). Such rigid compartmentalization encourages us to look for rational connections between spaces and objects and by deduction the mind that arranged them.\n\nThe Crypt with Setiâs sarcophagus is on the left, the picture gallery on the right (Soane, 1835-1836: Plate XXV).\n\nInstead, if we collapse the internal boundaries we can approach Soaneâs house-mind like the gyrifications of the cerebral cortex; where precise folding and wrapping packs a brain, like a parachute, into its brain-case. Scanning by magnetic resonance imaging (MRI) now reveals the extent of this folding for the hindbrain, the cerebellum. Compared to the neocortex the cerebellum is small, a tightly folded bundle of neuronal tissue with approximately 10 percent of the volume of the brainâs cortex. When unwrapped using MRI it becomes a two-dimensional, wafer-thin strip measuring 10cms by an astonishing 1metre (Sereno et al., 2020). The unwrapped cerebellum now grows exponentially in size achieving 78 percent of the total surface area of the neocortex. By comparison the macaqueâs cerebellum when unwrapped achieves only 33 percent.\n\nHuman evolution, it appears, has favoured growth in both the neocortex and the cerebellum. Folding, the process of gyrification, is therefore as important as relative brain size, a figure driven principally by the neocortex. The folding of the folia also provides another answer to the question, what kind of mind?\n\nA tightly swaddled cerebellum brings different areas of sensation into contact in a kaleidoscopic fashion; the example given by Sereno (2020) is a bit of lip found next to a chunk of the shoulder. As a result, the idea that the cerebellum primarily controls balance and movement needs to be augmented. In Serenoâs view it contributes not only to our five senses but also to pain, thought, and emotion. Its compact versatility contrasts with the neocortex where distinct lobes are associated with particular senses; vision in the occipital, sound in the temporal and so on (Carter, 2003).\n\nClearly there is much for neuro-imagers still to discover. What I take from these MRI images (Figure 3) is not just the physical folding of the brain, its gyrifications, but what folding affords for our metaphors of cognition. These MRI images provide a different embodied metaphor for the minds of deep history; one where wrapping and folding rather than size alone created relations and associations between people, stuff, and the world.\n\nThe image shows a Mercator transformation of the 3D space embedding a dense human brain tractography. The unfolding was performed from a ventral point of view and shows the arrival of white matter fibres to the neocortex. The cerebellum is at the top of the image at the midline. Immediately to the sides are the left and right temporal lobes, and the interhemispheric margin appears at the left and right borders of the image. Compiled by Katja Heuer and Roberto Toro https://wellcomecollection.org/works/trdzdrwk CC BY 4.0.\n\nFor the moment letâs investigate the implications for understanding Sir John Soaneâs Museum, and by extension any house. First, the house as a mind is a spatial suite of folded surfaces. It is never rigidly compartmentalized. Second, different times, spaces, materials, and people are brought into association through those folds. Bruno Latour (2005: 201) observes that time is always folded. He builds on Michel Serresâs (Serres and Latour, 1995) use of a crumpled handkerchief to show how a single surface of space and time can be changed by rubbing different spaces together. Such folding creates relations between things which are not always amenable to rational analysis. Time and space are best understood, like the unwrapped MRI brains (Figure 3), as folded folia. And from an archaeological perspective those folia wrap stuff.\n\nSoane arranged his collections in the spaces he designed to unite the three arts of architecture, painting and sculpture and as âstudies for my own mindâ (1835-6:vii). His marble bust, centrally positioned, is in visual contact with many classical antiques and it overlooks Setiâs sarcophagus in the basement (Figure 4). The millennia of time are folded within the space of his fixed gaze and the ambulatory perception of the visitor. Chronology did not interest him. His museum reminds me of John Phillips (1860: 51), President of the Geological Society, who declared that geological time âeluded the grasp of the imaginationâ. But he also described it as folded with this example: a gravel quarry in Oxfordshire, five metres deep, with mammoth bones at its base and the footprints of Charles Ist fleeing the Parliamentary army at the top. In a single stratified section recent and deep history touched, concertinaed into each other (Gamble, 2021: 132). Time and space are also wrapped and folded in Soaneâs Museum, from the depths of Setiâs sarcophagus to his own portrait bust (Figure 4).\n\nPhoto Clive Gamble.\n\nSoaneâs museum is a projection of the MRI kaleidoscope of the cerebellum as described by Serreno. Lips touch shoulders, compassion nestles against pain. Rather than a MINDâS EYE organizing everything it is instead a house where WALLS HAVE EARS.\n\nThis kaleidoscopic folding is on show in Soaneâs Picture Room; a small space crammed with paintings by Canaletto, Turner, and many others. The room is deceptive. It is a skin wrapped within a skin. The paintings are hung on folding doors, or planes, that open to reveal another layer on the reverse side. At one level this is a practical solution for a larger-than-the-space-available collection. But these first and second skins also fold time: in this case the fifteen ink and wash drawings by Giovanni Battista Piranesi (1720-1778) showing the ruined Greek temples at Paestum, in Italy (Thornton and Dorey, 1992: 41). These are hung alongside William Hogarthâs (1697-1764) eight paintings of A Rakeâs Progress with its different take on decline and decay. Piranesi shows the collapse of civilised glory in the face of time, while for Hogarth itâs the rapid ruination of a young man, Tom Rakewell; the history of a civilisation contrasted with a single biography. Time and space are always wrapped, their folia folded to make new patterns, fresh associations.\n\nThe ingrained container-habitus of many archaeologists studying cognitive evolution led Chris Gosden (2010: 40) to announce the death of such a mind to be replaced with the indivisible trinity of bodies, brains, and world (DeMarrais et al., 2004; Knappett, 2005; Dunbar et al., 2010; Conneller, 2011; Malafouris, 2013; Spikins, 2015). The mind that emerges from this trinity extends beyond our self-contained borders; our skins, clothes, kin, and houses, so that as Andy Clark (1997; Clark and Chalmers, 1998) states, âeverything leaksâ. As a result, cognition is porous and the agency arising from mentalizing walks a three-way street between brains, stuff, and the world we inhabit.\n\nThis understanding of cognition is expressed in the embodied metaphor MIND IS DISTRIBUTED. If mind is extended in this way, then the concept breaks free from the familiar container-habitus that guides how we think we think. Boundaries are replaced by flows and the solid partitions between categories such as reason and emotion collapse to resemble instead the folia in the tightly folded cerebellum (Figure 3).\n\nSoaneâs Museum is a mind distributed in time and through spaces. Thomas (2018) recounts how Soane extended himself beyond his death. He placed three time-capsules in his house and instructed his trustees to open them in 1866, 1886, and 1896. The contents of these sealed locations were regarded by the curators as a disappointment; among them newspapers, lottery tickets, stubs of cheque-books, bills, theatre tickets, notes from the spies he hired to follow his eldest son, and a pair of false teeth. In short no hidden masterpieces or even the key to the mysteries of the house and its contents. Simply the ephemera of a life lived. Thomas (2018: 138) draws a parallel with the 610 cardboard storage boxes that Andy Warhol filled on a daily basis with the mundane stuff that crossed his desk (https://www.warhol.org/timecapsule/andy-warhols-time-capsule-21/). These containers may be sealed like any tomb so that at first sight Warholâs boxes and Soaneâs time capsules pack neatly into the container-habitus to produce a familiar mind arising from âboxed-up livesâ. But that is because we are trained to see the walls of any container as impermeable; skin, for example, contains our inner selves, while clothes present outward identities; a case of âthereâs the essential Warhol in a boxâ, or âLetâs find the real Soane in a locked drawerâ. The distributed âWarholâ and âSoaneâ are not, however, neatly bounded entities with discrete minds. Although the artist and the architect both used containers to box their lives, they could not obscure the flows of agency between the trinity of brains, stuff, and the world. Instead, what they created transcends such embodied experience.\n\nIn his influential study of the prehistoric mind Steven Mithen (1996: 65-72) uses the container metaphor of church architecture to explain the process. Around the central nave of generalized intelligence were built four side chapels of specialized knowledge; technical, linguistic, social, and natural history. At some point the separate knowledge in these chapels was integrated by the evolution of cognitive fluidity. The result was a disembodied, super intelligence that eventually would write Descartesâs Regulae and drive the driverless car.\n\nFrom the perspective of the distributed mind, I would offer a different metaphor, an ARCHITECTURE WITHOUT WALLS, to counterbalance the slowly evolving cathedral. Houses serve as proxies for minds not because of what they contain, or how many rooms they have, but how they mediate the flows of agency between brains, bodies, and the world. Houses, like brains, can vary in size and complexity from Diocletianâs palace to an igloo. Brain size increases during hominin evolution and particularly during the Middle Pleistocene, 800,000 â 125,000 years ago (Gamble et al., 2014; Pope et al., 2018). Philip Rightmire (2004) identifies an increase of c. 20 per cent in brain size and encephalisation quotients between 0.6-0.2 million years ago. This significant growth in such an energetically expensive tissue (Aiello and Wheeler, 1995) is not however matched by a comparable shift in stone, or any other hominin technology, and certainly not in containers. The changes are instead to be found in the persistent use of places and landscapes (Shaw et al., 2016) that has been described by Matt Pope (2018) as crossing a threshold in hominin evolution.\n\nContainer artefacts, most notably hearths (Alperson-Afil and Goren-Inbar, 2010; Gowlett, 2010, 2016; Karkanas et al., 2007; Zhou et al., 2012; MacDonald et al., 2021), are present both before and during this time of increasing brain size. But houses are not (Kolen, 1999). There have, of course, been claims for substantial structures. At Olduvai Gorge, site DK Level 3, Mary Leakey (1971: Fig. 7) interpreted a circular agglomeration of stones with a maximum diameter of 4.3 metres as the base of a hut. Richard Potts (1988), however, interprets this 1.9 million year old accumulation as a stone cache.\n\nFrom the Middle Pleistocene of Europe, the multiple long huts with hearths at the coastal locale of Terra Amata, France (de Lumley and Boone, 1976; de Lumley, 2009) (Figure 5) have not stood up to detailed scrutiny (Villa, 1976-1977, 1982). The Bilzingsleben huts in Germany (Mania, 1990, 1991) are part of a fan deposit subsequently criss-crossed by small geological faults. The geomorphological setting questions the claims for good preservation that are essential to the interpretation of dwellings (Gamble, 1999: 153-163). On the other hand, Middle Pleistocene locales with exceptional preservation such as Schöningen, Germany (Conard et al., 2015) and Boxgrove, England (Pope et al., 2020) lack evidence such as post-holes for structures. As Jan Kolen (1999: 162) concluded more than 20 years ago, the evidence from Europe is most economically interpreted as centrifugally produced living structures rather than architecture. A pattern that results from the activities of bodies using space. An example of this patterning is the âcabinâ inside Lazaret Cave, France (de Lumley et al., 2004).\n\n21 living floors with huts ranging in length from 8 to 15 metres have been identified (de Lumley, 2009) photo Clive Gamble.\n\nKolen also observed that the first 10,000 years of the European Upper Palaeolithic (c. 40-30,000 years ago) mostly lacks durable architecture and unambiguous burials with grave goods. These start no earlier than the Pavlovian (32-30,000 years ago) of the Czech Republic and extend eastwards into Ukraine and Russia. Even so, the evidence for huts and houses remains contentious. For example, at Kostenki 11, Russia (Figure 6) recent excavations of a mammoth bone structure have shown, that whatever else it is, it is not a house (Pryor et al., 2020).\n\nThe structure is 12.5 metres in diameter and is the third found at K11. It is dated to 25,000 years ago (Pryor et al., 2020). Photo courtesy of Alexander E. Dudin.\n\nThis brief survey of artificial structures in deep history reminds us that the most potent practical metaphor for a mind, the house, is absent. The evidence, of course, is biased towards Europe. Houses older than 30,000 years may yet be found in Africa and Asia. But by comparison hearths, that form such a significant feature in many houses, are found throughout the Old World in the Lower and Middle Pleistocene (Gowlett, 2016).\n\nWe are indeed confronted by an ARCHITECTURE WITHOUT WALLS where spatial patterning exists but without being artificially contained. A momentâs reflection suggests this is unsurprising. Searching for archaeological evidence for houses betrays our container-habitus when it comes to organising and investigating the past. But hominins have always authored spaces to enact the varied performances of social life. One such example is the horse butchery locale at Boxgrove, 500,000 years ago. Matt Pope et al. (2020, Pope, 2020) have shown how the evidence reveals the minute-by-minute movement and activities of a tight-knit group; a community of people, young and old, working together in a co-operative and highly social way. From the flint and bone evidence they propose a group of at least 30-40 people (Figure 7).\n\nArtist Lauren Gibson sets the scene of the horse butchery site, GTP-17. A performance space is created by the ring of people involved in an activity that took place one day, half a million years ago. Reproduced with the permission of Lauren Gibson and UCL Institute of Archaeology and © UCL 2020. CC BY-NC-ND licence.\n\nThe patterning of the artefacts in front of the Boxgrove cliffs suggests that people formed a ring around the horse carcass, an area of about 90 square metres. Here was a temporary container of social life, an ARCHITECTURE WITHOUT WALLS formed by the participants in a gathering. The architecture of this event needed neither partitions nor hearths and certainly none of Gellâs gaudy skins that dominate Soaneâs Museum. It is an image of the distributed mind, recovered by archaeologists from the fragments of deep history. Hominins have always gathered. Social life has always been performed within porous containers as bodies shifted in the ring to get a better view.\n\n\nConclusion: beyond big brains\n\nHominin minds have always existed irrespective of either the size of the neo-cortex in their skulls or if they built and lived in houses. In this paper I have argued that linking significant changes in hominin cognition such as language, planning, and memory to the evolution of big brains is an instance of our own boxed-up thinking applied to hominin evolution. We need to move beyond a deep history conditioned by an unrecognised container-habitus where minds are compartmentalized by the artefacts we think with and through, without thinking.\n\nA first step is to recognise hominin cognition as distributed between stuff, brains, and the world. The stuff I have examined here are houses and I started with Gellâs statement that to enter a house is to enter a mind, a sensibility. That is patently the case with the houses of Mesolithic Lepenski Vir (BoriÄ, 2016), the rich house ethnographies of Africa (Denyer, 1978) and North America (Morgan, 1881), the ephemera of nomad tents (Cribb, 1991: Chapter 7), and the properties currently for sale on Rightmove and Zoopla. Sir John Soaneâs Museum is an eccentric example but makes Gellâs (1998: 251-8) case as effectively as his chosen example of a Maori meeting house.\n\nThe lack of credible houses before 30,000 years ago does not signify there were no minds. The evolution of larger more complex houses since then does not suggest a concomitant increase in the cognitive power of those who built and lived in them. So why should the evolution of bigger brains necessarily suggest, like building a cathedral, either more complex or different minds?\n\nBrain size can be used to predict growth in an individual homininâs network of relations, while the evolution of language is part of the story of a social brain hypothesis where the benefits of social life selected for encephalisation (Dunbar, 1998; Dunbar et al., 2010; Gamble et al., 2014). But as James Cole (2015) has pointed out, increasing brain size among hominins reveals possibilities for the cognitive underpinnings of social life, not what was realised. Only archaeology can do that, because only archaeology can access the varied scales of time and place to write the narrative of humanity. Brain size is one factor and by considering it we have moved forward our understanding of hominin mentalising as an evolutionary process (Gamble et al., 2011; Gowlett et al., 2012). But brain size now needs to be complemented by the folding of the folia and the associations that they allow and which we are only just beginning to appreciate.\n\nDeep history has never been a foreign country, the Originsland I have written about elsewhere (Gamble, 2007), unless it is viewed through the lens of the container-habitus. What binds us to deep history is a distributed mind and an ARCHITECTURE WITHOUT WALLS that guides social life. That is not to say that change didnât occur. It did. At some point in humanityâs story containers, as exemplified by houses, came to do our thinking for us. Understanding why that happened remains one of the great challenges for deep history. And to answer that we need to unwrap the brain.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgments\n\nI am grateful to the editors for inviting me to write for this collection and to William Davies and Matt Pope for discussions about huts and performances in deep history. Lauren Gibson and Louise Rayner kindly supplied the original artwork for the Boxgrove gathering.\n\n\nReferences\n\nAiello L, Wheeler P: The expensive-tissue hypothesis: the brain and the digestive system in human and primate evolution. Curr. Anthropol. 1995; 36: 199â221.\n\nAlperson-Afil N, Goren-Inbar N: The Acheulian site of Gesher Benot Yaâaqov volume II: ancient flames and controlled use of fire. Dordrecht: Springer; 2010.\n\nBoriÄ D: Posmrtni obredi na Lepenskom Viru: obrasci pogrebne prakse: iskopavanja Dragoslava SrejoviÄa = Deathways at Lepenski Vir: patterns in mortuary practice: excavations of Dragoslav SrejoviÄ. Beograd: Srpsko arheeloÅ¡ko druÅ¡tvo = Serbian Archaeological Society; 2016.\n\nCarter R: Mapping the mind. London: Phoenix; 2003.\n\nChilde VG: What happened in history. Harmondsworth: Penguin Books; 1942.\n\nClark A: Being there: bringing brain, body and world together again. Cambridge, MA: MIT Press; 1997.\n\nClark A, Chalmers DA: The extended mind. Analysis. 1998; 58: 7â19. Publisher Full Text\n\nCole J: Handaxe symmetry in the Lower and Middle Palaeolithic: implications for the Acheulean gaze. Settlement, society and cognition in human evolution: Landscapes in mind. Coward F, Hosfield R, Pope M, et al., editors. Cambridge: Cambridge University Press; 2015; pp. 234â257.\n\nConard NJ, Serangeli J, Böhner U, et al.: Excavations at Schöningen and paradigm shifts in human evolution. J. Hum. Evol. 2015; 89: 1â17. PubMed Abstract | Publisher Full Text\n\nConneller C: An archaeology of materials: substantial transformations in early prehistoric Europe. 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Publisher Full Text\n\nGamble CS, Gowlett JAJ, Dunbar R: Thinking big: the archaeology of the social brain. London: Thames and Hudson; 2014.\n\nGell A: Art and agency: towards a new anthropological theory. Oxford: Clarendon Press; 1998.\n\nGibson JJ: The ecological approach to visual perception. Hillsdale: Erlbaum Associates; 1979.\n\nGosden C: The death of the mind. The cognitive life of things: recasting the boundaries of the mind. Malafouris L, Renfrew C, editors. Cambridge: McDonald Institute for Archaeological Research; 2010; pp. 39â46.\n\nGowlett JAJ: Firing up the social social brain. Social brain and distributed mind. Dunbar R, Gamble C, Gowlett JAJ, editors. Oxford: Oxford University Press; 2010; pp. 341â366.\n\nGowlett JAJ: The discovery of fire by humans: a long and convoluted process. Philos. Trans. R. Soc. B. 2016; 371: 20150164. 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Leiden: University of Leiden and European Science Foundation; 1999; pp. 139â175.\n\nLakoff G, Johnson M: Philosopy in the flesh: the embodied mind and its challenge to western thought. New York: Basic Books; 1999.\n\nLatour B: Reassembling the Social: An introduction to Actor-Network-Theory. Oxford: Oxford University Press; 2005.\n\nLeakey MD: Olduvai Gorge: excavations in Beds I and II 1960-1963. Cambridge: Cambridge University Press; 1971.\n\nMacDonald K, Scherjon F, van Veen E , et al.: Middle Pleistocene fire use: The first signal of widespread cultural diffusion in human evolution. Proc. Natl. Acad. Sci. 2021; 118(31): e2101108118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMalafouris L: How things shape the mind: a theory of material engagement. Cambridge Massachusetts: MIT Press; 2013.\n\nMania D: Auf den Spuren des Urmenschen: Die Funde von Bilzingsleben. Berlin: Theiss; 1990.\n\nMania D: The zonal division of the Lower Palaeolithic open-air site Bilzingsleben. Anthropologie. 1991; 29: 17â24.\n\nMithen S: The prehistory of the mind. London: Thames and Hudson; 1996.\n\nMorgan LH: Houses and house-life of the American Aborigines. Washington: Government printing Office; 1881.\n\nMurdoch D, Cottingham J, Descartes R, et al.: Rules for the Direction of the Mind. Cambridge: Cambridge University Press; 1985.\n\nPhillips J: Anniversary address of the President. Q. J. Geol. Soc. Lond. 1860; 16: xxviiâlv. Publisher Full Text\n\nPope M: Thresholds in behaviour, thresholds of visibility: landscape process, assymmeteries in landscape records and niche construction in the formation of the Palaeolithic record. Crossing the human threshold: dynamic transformation and persistent places during the Middle Pleistocene. Pope M, McNabb J, Gamble C, editors. London: Routledge; 2018; pp. 24â39.\n\nPope M: Butchering a horse at Boxgrove. Br. Archaeol. 2020; 175: 18â29.\n\nPope M, McNabb J, Gamble CS: Crossing the human threshold: dynamic transformation and persistent places during the Middle Pleistocene. London: Routledge; 2018.\n\nPope M, Parfitt S, Roberts M: The horse butchery site: a high resolution record of Lower Palaeolithic hominin behaviour at Boxgrove, UK. UCL: Spoil Heap Publications; 2020; p. 23.\n\nPotts R: Early hominid activities at Olduvai. New York: Aldine; 1988.\n\nPryor AJE, Beresford-Jones DG, Dudin AE, et al.: The chronology and function of a new circular mammoth-bone structure at Kostenki 11. Antiquity. 2020; 94(374): 323â341. Publisher Full Text\n\nRenfrew C: Before civilization. London: Johnathon Cape; 1973.\n\nRenfrew C: The sapient behaviour paradox: how to test for potential? Modelling the early human mind. Mellars P, Gibson K, editors. Cambridge: McDonald Institute for Archaeological Research; 1996; pp. 11â14.\n\nRightmire GP: Brain size and encephalization in early to Mid-Pleistocene Homo. Am. J. Phys. Anthropol. 2004; 124(2): 109â123. Publisher Full Text\n\nSereno MI: The Unfolded Cerebellum Is Three Feet Long: Technology Networks Neuroscience News and Research.2020. Reference Source\n\nSereno MI, Diedrichsen J, Tachrount M, et al.: The human cerebellum has almost 80% of the surface area of the neocortex. Proc. Natl. Acad. Sci. 2020; 117(32): 19538â19543. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSerres M, Latour B: Conversations on Science, Culture, and Time. Ann Arbor: University of Michigan Press; 1995.\n\nShaw A, Bates M, Conneller C, et al.: The archaeology of persistent places: the palaeolithic case of La Cotte de St Brelade, Jersey UK. Antiquity. 2016; 90: 1437â1453. Publisher Full Text\n\nSoane J: Descriptions of the House and Museum on the north side of Lincolnâs Inn Fields, the residence of Sir John Soane. London: Privately printed by Levey, Robinson, and Franklyn, St. Martinâs Lane; 1835-1836.\n\nSpikins P: How compassion made us human: the evolutionary origins of tenderness, trust and morality. Barnsley: Pen and Sword; 2015.\n\nThomas S: A âstrange and mixed assemblageâ: Sir John Soane, Archivist of the Self. Stud. Romant. 2018; 57(1): 121â142. Publisher Full Text\n\nThornton P, Dorey H: A miscellany of objects from Sir John Soaneâs Museum: consisting of paintings, architectural drawings and other curiosities from the collection of Sir John Soane. London: Laurence King; 1992.\n\nTilley C: Metaphor and material culture. Oxford: Blackwell; 1999.\n\nVilla P: Sols at niveaux dâhabitat du paleolithique inferieur en Europe et du Proche Orient. Quaternaria. 1976-1977; 19: 107â134.\n\nVilla P: Conjoinable pieces and site formation processes. Am. Antiq. 1982; 47: 276â290.\n\nZhou Z, Guan Y, Wang C, et al.: Remains of human fire-use: an overview of paleolithic hearth and human fire-use behavior. Acta Anthropologica Sinica. 2012; 31(1): 24â40."
}
|
[
{
"id": "168519",
"date": "03 May 2023",
"name": "John Chapman",
"expertise": [
"Reviewer Expertise My specialisation as primarily a âNeolithicâ archaeologist means that I am much more at home in the container-habitus than in an architecture without walls",
"although I have a good awareness of the latest trends in Palaeolithic archaeology."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nClive Gambleâs opinion piece portrays a dazzling combination of disparate ideas enlisted to make the case for closer links between recent developments in the science of the brain and developments âout thereâ in the world, whether concerning time, buildings, landscapes or social structures. We cannot but admire the range of thoughts that Gamble cites in support of his key metaphor that the folding of folia in the cerebellum stands for growing networks, increasing social complexity and developing bodily skills in the Palaeolithic. Interleaved with Latourâs notion that time is always folded and Gellâs idea that âto enter a house is to enter a mindâ, we have Gosdenâs trinity of bodies, brains and the world connected by the flow of agency, as well as Gambleâs own research on the expanding use of containers â whether settlements, houses or even minds. These thoughts give rise to other ideas, principally in the form of metaphors. Indeed, this opinion piece is brimming over with metaphors, which range from six separate if interlinked embodied metaphors (\"the mindâs eye\"; \"the walls have ears\"; \"the distributed mind\"; \"the mind is a container of ideas\"; \"ideas are objects\"; \"knowing is seeing\") to two metaphors for hunter-gatherer settlement â Mithenâs church architecture and the authorâs \"architecture without walls\" â and finally a metaphor for later developments â the \"container-habitus\". This shimmering metaphorical surface shines and sparkles, working its magic on the reader much in the form of Gellâs dazzling canoe-prow that engendered better exchange rates for Melanesian mariners. There is time and space here to scrutinize more closely only the change from one metaphor to another â how the Neolithic container-habitus developed out of, or in contradistinction to, the Palaeolithic and Mesolithic âarchitecture without wallsâ. This is the great container shift which Gamble seeks to explore. I offer a brief commentary here.\nThere is a consensus among Palaeolithic specialists that no houses were built before 30,000 years ago â at least in Europe. Instead, many spaces were authored to enact performances of social life, such as the horse butchery place at Boxgrove, dated to c. 500,000 years BP. One of Gambleâs most intriguing links is between the increasing brain size of hominins between 600,000 and 200,000 years BP, and their persistent use of the same places and landscapes. The slow increase in the importance of place-based and landscape-based identities for these hunter-gatherers stimulated the emergence of spatial differences between favoured and non-favoured places, and between special landscapes and other zones. The concentration of people and their objects at favoured places promoted deepening practical and emotional links which could only become deeper with ritual sanctification of practices and/or of the places themselves. But the seasonal nature of such congregations left unsolved the essential problem of Palaeolithic socialization â the missing people who were absent from their seasonal mates for most of the year. With population densities of between 0.05 and 0.005 persons per km2, such absence was the norm in territories of many thousands of km2 and inevitably created social issues. The appropriate Palaeolithic metaphor would be Cousin Antonio, whose absence from the home base sparked conversations such as âWhere is Antonio? I havenât seen him since last summer. I hope heâs OK.â\nLong ago, Martin Wobst suggested that open, overlapping mating networks characterized the Palaeolithic until near the end of the Last Glacial period, when locational constraints, selection for communal tasks and increasing population densities led to the emergence of new forms of mating network. In Gamblian terms, this was a new form of container â the closed mating network. The flip side of a declining momentum for seasonal mobility was the pleasure-and-pain of a more sedentary way of life, in which comments about Cousin Antonio had changed to: âWhat can we do about Antonio? He snores all night and sings all day.â Not only was it vital to contain Antonio in a building but the building also contained the collections of objects made by, or traded for, the household. The possibility that the household could eat and drink in a new state of privacy meant the establishment of closer, countervailing ties with those outside the private space â the neighbours â in a new form of shared hospitality, with its own social dynamic and multiplier effect. Furthermore, a building could echo, symbolize or memorialize not only associated people, animals or fish but also other significant features of the landscape, such as the trapezoidal mountain of Treskavec whose shape was materialized in the house-plans of Lepenski Vir. No wonder that the Neolithic soon became, in DuÅ¡an BoriÄâs striking phrase, âthe age of buildingâ. Gamble is correct to assert that the container-habitus â the house â did Neolithic personsâ thinking for them.\nWhat I cannot comment on directly is how increased levels of wrapping and folding in the cerebellum interacted with âout-of-bodyâ experiences to create relationships and associations between brains, people, stuff and the world. But it is a provocative metaphor, which will surely lead to new ways of conceptualizing the human condition. For this, we owe much gratitude to Clive Gamble.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly",
"responses": []
},
{
"id": "180841",
"date": "21 Aug 2023",
"name": "Andy Jones",
"expertise": [
"Reviewer Expertise Prehistoric art and material culture (later prehistoric and ethnographic)."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is vintage Gamble! Erudition and Big ideas covering large expanses of prehistory, presenting us with a whole new vision of the Palaeolithic and later periods. This paper is sure to be influential and I recommend that it is indexed verbatim (i.e. no corrections).\n\nThere are a few things I think the author could think about for improving the paper (for future development):\nI was curious that the concept of 'Distributed Minds' was used without mentioning one of Gell's prime examples: the Maori meeting house. This seems to be relevant to the argument presented here.\n\nFolding. It might be worth looking at Gavin Lucas' Making Time book which develops the scrumpled tissue metaphor for time. Also worth looking at Gilles Deleuze on the fold - this is where Latour stole the idea from!\n\nThe critique of archaeologists as possessing the container-habitus is well taken, but I wondered how this might relate to the Wengrow-Graeber 'Dawn of Everything' thesis? Here Wengrow and Graeber seem to be offering a more fluid understanding of early societies from the Palaeolithic to Neolithic. It might be worth addressing in future. Does this connote an 'architecture without walls'?\n\nIt is a shame that the story ends in the upper Palaeolithic. Continuing the story into the Mesolithic and Neolithic will probably pay dividends - particularly as we encounter a proliferation of evidence for houses in these later periods. It might be worth looking into Ian Hodder's concept of 'History houses' at Catal Hoyuk in this regard. Also Richard Bradley's recent publications on houses.\n\nFinally, how do we regard caves? Surely these are containers too? The thesis developed here helps us think afresh about Cave art, and the (relatively scant) evidence for settlement and burial in Cave contexts. Does the container-habitus begin with the cave, and become transferred to built architecture at a later date? Was the architecture without walls first generated in places with natural walls (making the walls redundant), allowing for distributed minds to unfold outside the cave?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-301
|
https://f1000research.com/articles/12-298/v1
|
17 Mar 23
|
{
"type": "Research Article",
"title": "Identification of potential compounds for the management of multidrug-resistant tuberculosis using computational methods",
"authors": [
"Winnie Gatwiri",
"Richard Kagia",
"Richard Kagia"
],
"abstract": "Background: Tuberculosis is caused by Mycobacterium tuberculosis and is spread through the air. Multidrug-resistant tuberculosis (MDR TB) has become a global health concern. This study focuses on developing alternative compounds to Levofloxacin, Moxifloxacin, Bedaquiline, Kanamycin, Amikacin, Cycloserine, Ethambutol, Pyrazinamide, Linezolid and Streptomycin that can be used to treat patients with multidrug resistance TB. The virtual screening will aid in discovering other possible compounds for use in the management of MDR TB, thereby providing a superior alternative to currently existing medications and aid in eradicating TB. The objective of this study was to identify potential compounds that can be used in managing MDR TB in chronic tuberculosis patients using computational methods. Methods: The Swiss Similarity tool was used to identify similar compounds to the tuberculosis drugs in a ZINC database. Compounds more similar to the tuberculosis drugs were selected and used to test the molecular docking with their respective targets. The pharmacokinetics and toxicity profiles of the selected compounds were analyzed using Swiss ADME and Pro Tox Server, respectively. Results: Overall, 90 compounds had higher binding energies than the medications, 88 had lower binding energies, and 14 had binding energies that were equivalent to those of the drugs. Only 14 of the 200 compounds lacked CYP inhibition, were p-glycoprotein substrates, had superior docking scores to the compounds, and fell into toxicity classes V and VI. Conclusions: The 14 potential compounds should undergo further in vivo and in vitro studies to develop new compounds for managing multidrug-resistant tuberculosis.",
"keywords": [
"ZINC database",
"SwissADME",
"MDR TB",
"fatty acid synthetase",
"ligand-based virtual screening."
],
"content": "Introduction\n\nAccording to World Health Organization (WHO, 2021), tuberculosis is caused by Mycobacterium tuberculosis and is spread through the air. Mycobacterium tuberculosis is thought to infect a quarter of the population and is more dangerous in malnourished people (WHO, 2021). A Mycobacterium strain resistant to at least Rifampicin and Isoniazid, the first-line medications used to treat all tuberculosis patients, causes multidrug drug-resistant TB (CDC, 2016). According to a study that was done by Seung et al., in 2015, it was found that multidrug-resistant TB has increased and has become a global health concern. MDR-TB affects over 470.000 people and kills roughly 180,000 yearly (WHO, 2021).\n\nMost African countries still have gaps in diagnosis and treatment, resulting in a rise in MDR-TB patients. Only 68% of the individuals were found to have MDR-TB. MDR TB has emerged as a problem in Sub-Saharan Africa. The increase in MDR TB is due to a scarcity of appropriately trained health staff and resources for tuberculosis control (Ismail et al., 2018). In 2016, MDR TB was found to be 1.5% prevalent in Sub-Saharan Africa, with MDR cases increasing five times in Ethiopia for patients previously treated for TB compared to new cases (Musa et al., 2017). According to a recent study carried out in Mbagathi and Chandaria in 2015 and 2016, the prevalence of isoniazid resistance in Kenya was 0.8%, and 0.8% for multidrug resistance (Ogari et al., 2019). There has been less research in Kenya to illustrate the frequency of MDR TB. Therefore, there is insufficient data on the disease. There are also limited diagnostic tools, so detection is poor (Ombura et al., 2016).\n\nThe method of looking via computer databases for potentially active substances is known as virtual screening. There are two categories of virtual screening: ligand-based drug design and target-based drug design. Ligand-based drug design searches databases for compounds comparable to known ligands with similar biological actions (Neves et al., 2018). Virtual screening aids in the discovery of alternative compounds that perform the same biological function as MDR TB drugs but are more effective and less harmful (Banegas et al., 2018).\n\n\nMethods\n\nThe study was carried out in August 2022. All of the medications were analyzed in silico. The canonical smiles of the drugs (pyrazinamide, ethambutol, cycloserine, bedaquiline, streptomycin, amikacin, kanamycin, linezolid, levofloxacin, and moxifloxacin) were obtained from PubChem (RRID:SCR_004284). The drugs were selected as they are the most common second-line agents that are used in the management of MDR TB. They are also easily found in hospitals.\n\nThe canonical smiles of the drugs were inserted into the Swiss Similarity online tool to identify other compounds that are similar to the drugs. Because it provides molecular shapes and properties, the Swiss similarity tool was used to determine molecular similarities. The combined technique of Swiss similarity that involves molecular fingerprints, pharmacophore recognition and shape-based similarity was used to screen the ZINC database of the drug-like compounds. Similarity scores were obtained based on the combination of Electro-shape 5D Manhattan distance and Tanimoto coefficient. 20 analogs that had the highest similarity scores to the reference compounds were selected and sketched on a PubChem sketcher, and the Molfile was downloaded and saved in an LBVS folder.\n\nThe drugs were downloaded in SDF format and saved in a drug-specific folder. Similar compound structures were relocated from the LBVS folder to the SBVS folder. The drugs and analogsâ Molfile were downloaded and transformed into their 3D structures using Avogadro version 1.1.0 software (Avogadro, n.d.) (RRID:SCR_015983). MMFF94S was then used to optimize the 3D structure to their most stable conformations.\n\nThe Chimera version 1.14c software (RRID:SCR_004097) was used to add hydrogen atoms and charge to the stable conformations of the compounds. The protein databank was then used to download the drugsâ respective target receptors (Fatty acid synthase, arabinosyltransferase, D-alanine L-racemase, F-ATP synthase subunit, 30S ribosomal RNA, 23S ribosomal RNA of 50S ribosomal subunit, DNA gyrase) which was saved in an SBVS folder. The Chimera software was used to remove the non-standard residues from the molecular target receptors and was saved as the final receptor. The surface binding of the selected compounds with the target receptors was done using the Auto dock vina (RRID:SCR_011958), found in the Chimera software. The surface binding of the drugs was also done to act as a positive control. Ligand interactions were then examined using discovery studio version 19.1.0.18287 software (RRID:SCR_015651).\n\nThe SWISS ADME online tool, which predicts drug and medicinal chemistry similarity and pharmacokinetic features, was used to forecast the pharmacokinetic profile of the compounds. The selected compoundsâ pharmacokinetic parameters were entered into the SWISS ADME, and the compoundsâ absorption, distribution, metabolism, and excretion properties were obtained.\n\nThe ProTox RRID:SCR_018506 server was utilized to predict the toxic levels of the medications and comparable substances. The canonical smiles of the drugs and the analogs were obtained from pubchem and pasted on ProTox server. Oral toxicity, immunotoxicity, carcinogenicity, cytotoxicity, and mutagenicity were all determined using the ProTox server. In determining oral toxicity, class 1 means itâs fatal if swallowed (LD50 less than 50 mg/kg), class II fatal if swallowed with an LD50 of between 5 mg/kg to 50 mg/kg, class III is toxic when swallowed with an LD50 of between 50 mg/kg to 300 mg/kg, class IV is harmful wen swallowed with and LD50 of between 300 mg/kg to 2000 mg/kg, class V may be harmful when swallowed with an LD50 of between 2000 mg/kg to 5000 mg/kg and class VI is non-toxic with an LD50 of greater than 5000 mg/kg.\n\n\nResults\n\nCycloserine had a docking score of -4.6, and the analogs had higher binding energies than cycloserine (Muturia, 2023). The analogs of cycloserine that were chosen had a predicted toxicity class of class V as shown in Table 1, which indicated that they were less hazardous. All substances lacked CYP1A2 inhibition, CYP3A4 inhibition, CYP2D6 inhibition, CYP2C19 inhibition, BBB penetration, and a p-glycoprotein substrate. Except for ZINC3906759, all substances were anticipated to have a high GI absorption. ZINC9592220, ZINC95922219, and ZINC39067759 were predicted to be active for mutagenicity and carcinogenicity. The interaction of cycloserine analog ZINC39067759 with D-alanine is due to van der Waals, unfavorable charges and attractive charges that indicate possible ligand and protein interactions as shown in Figure 1.\n\nSource: this study. The diagram was created by discovery studio software.\n\nThe docking score for amikacin was 5.5. In comparison to amikacin, two of the chosen analogs had binding energies that were the same, one had greater binding energy, and seven had lower binding energies. Amikacin did not follow the Lipinski rule of five. Amikacin belonged to class V, while the chosen analogs were in classes V and VI as indicated in Table 1. All of the substances demonstrated minimal GI absorption and no CYP1A, CYP2C9, CYP2C19, or CYP3A4 inhibition. Except for ZINC44401823, all substances had a p-glycoprotein substrate. The interaction of amikacin with the 30s ribosomal subunit is due to van der Waals forces, carbon-hydrogen bonds, unfavorable positive-positive charges as well as conventional hydrogen bonds as shown in Figure 2.\n\nSource: this study.\n\nThe binding energies of the analogs were higher than that of pyrazinamide, which had a binding energy of -4.9. Except for ZINC95709439 and ZINC00351669, which are in toxicity class V, all compounds had a predicted toxicity level of IV as shown in Table 1. None of the substances inhibited CYP1A2, CYP2C9, CYP3A4, or CYP2C19, permeated the blood-brain barrier, or served as a p-glycoprotein substrate. Additionally, their GI absorption was high. Both ZINC95709439 and ZINC06524479 were active for hepatotoxicity and mutagenicity, respectively. Mutagenicity and immunotoxicity were both active for ZINC66351878 and ZINC61508077, respectively. The interaction of pyrazinamide and fatty acid synthetase is due to pi-alkyl, conventional hydrogen bond, and van der Waals forces as shown in Figure 3.\n\nSource: this study.\n\nEthambutol had a binding energy of -4.7, and all of its analogs had greater binding energy. All substances lacked BBB penetration, p-glycoprotein substrate, CYP1A2 inhibition, CYP2C19 inhibition, CYP2C9 inhibition, and CYP3A4 inhibition. Ethambutol belongs to toxicity class IV. As indicated in Table 1, the predicted toxicity class for four of the analogs was V, whereas the predicted toxicity class for the other six analogs was VI. Except for ZINC23477159, ZINC19364558, ZINC2247967, and ZINC22947968, all compounds showed a high GI absorption. The carcinogenicity of ZINC19365151, ZINC19364877, ZINC19361997, and ZINC100648389 and ZINC19365150 was active. The interaction of ethambutol and arabinosyl transferase is due to van der Waalâs forces, attractive charges, conventional hydrogen board, carbon-hydrogen board, alkyl, pi-alkyl, and unfavorable positive-positive charges as shown in Figure 4.\n\nSource: this study.\n\n\nDiscussion\n\nThe SwissSimilarity tool was used to find substitute compounds that were similar to the medications used to treat MDR TB. From each medication, 20 analogs were chosen. The 20 analogs that were selected were based on the highest similarity scores to the reference compounds. The comparison of the docking energies was used to identify compounds with the highest docking energies as these are the compounds with the best affinity to the receptors. Out of the 200 analogs chosen, 98 had higher binding energies, 88 had lower docking scores, and 14 had the same docking scores as the primary medication. 98 compounds would have been chosen because they had the best docking scores if we had only looked at docking scores. 20 of the 98 analogs were for pyrazinamide, 20 were for cycloserine, and 20 were for ethambutol. Meaning all of their analogs had the best docking scores and hence are potential compounds that could undergo further studies compared to the other compounds.\n\nAccording to the Lipinski rule of drug-likeness, the analogs of bedaquiline, cycloserine, moxifloxacin, ethambutol, pyrazinamide, levofloxacin, and linezolid all had molecular weights less than 500, hydrogen acceptors less than 10, log P less than 5, and hydrogen bonds less than 5, making them all orally active (Daina et al., 2017). Since they did not adhere to the Lipinski rule, streptomycin, amikacin, kanamycin, their analogs, and bedaquiline are not orally active (Daina et al., 2017). Class I toxicity have extremely lethal effects when ingested and has an LD50 of less than 5 mg/kg. Class III toxicity has an LD50 range of 50 mg/kg to 300 mg/kg and may be hazardous when ingested, while class II toxicity has an LD50 that ranges from 5 mg/kg to 50 mg/kg and are lethal when ingested. Class IV has a potential for harm when ingested and has an LD50 range of 300 to 2000 mg/kg. Class V is less dangerous and can be consumed by humans without having negative effects because it has an LD50 value of more than 2000. Class VI is non-toxic and has an LD50 of more than 5000, therefore humans can consume it without experiencing any negative consequences (Gadaleta et al., 2019).\n\nCycloserine works by preventing the peptidoglycans from forming, which weakens the cell wall and causes the bacteria to die. When used orally, it has a high GI absorption of between 70% and 90% (Wishart et al., 2018). It is necessary to lower the doses for people who have renal toxicity (Goodman & Gilman, 2018). Cycloserine has several negative side effects, including anxiety, mental distress, nervousness, nightmares, muscular twitching, and vertigo (Wishart et al., 2018). Consuming cycloserine with fattening foods reduces its absorption (Wishart et al., 2018).\n\nAmikacin, generated from kanamycin A, attaches to the 30S ribosomal subunit and prevents mRNA from binding to it and tRNA from accepting it, preventing bacterial growth (NCBI, 2022a). When injected intramuscularly, it has a high absorption rate, but topical and oral absorption is poor (NCBI, 2022a). It is eliminated by the kidney and may cause neuromuscular blockade, nephrotoxicity, and ototoxicity as side effects (Wishart et al., 2018).\n\nAccording to National Center for Biotechnology Information (2022b), pyrazinamide inhibits fatty acid synthetase 1, which prevents the bacteria from producing the fatty acids necessary for growth and replication, leading to the death of the bacteria. It enters the GI system quickly and is 10% bound to plasma proteins. At least 70% of the oral dose is eliminated in the urine, primarily by glomerular filtration, and it is subject to hepatic metabolism (NCBI, 2022b). Adverse effects may be stomach upset, fatigue, easy bruising, skin rash, yellowing of the skin or eyes, liver injuries, arthralgia, malaise, and urticaria. When taken with abacavir, pyrazinamide may reduce the drugâs elimination, which could raise the drugâs serum levels and cause toxicity (Wishart et al., 2018). Acetaminophen and Aceclofenac may reduce the elimination rate of pyrazinamide, increasing its concentration and causing toxicity (Wishart et al., 2018).\n\nEthambutol, a bacteriostatic drug, was created to treat Mycobacterium tuberculosis strains that were resistant to the antibiotic isoniazid. It prevents the synthesis of cell wall constituents like arabinogalactan and lipoarabinomannan by inhibiting arabinosyltransferases. Inhibiting the production of lipoarabinomannan prevents mycobacterial cells from interacting with the host cells. It has an oral bioavailability of between 75% and 80% (Wishart et al., 2018).\n\nEthambutol has a plasma binding of 20% to 30% and is metabolized by aldehyde dehydrogenase. Ethambutol is eliminated in urine in two forms: unmodified in 50% of cases and inactive metabolites in 8% to 15% of cases. Optic neuropathy, joint discomfort, pruritus, abdominal pain, malaise, and dizziness are some undesirable symptoms that may be noticed. Antacids shouldnât be taken with the medication because they will stop Ethambutol from being absorbed (Wishart et al., 2018).\n\n\nConclusion\n\nThe discovery of new pharmaceuticals that may be utilized to treat multidrug-resistant tuberculosis has been made possible by this researchâs contribution to the identification of compounds with higher docking scores than conventional treatments. Patients with other comorbidities will benefit from compounds without cytochrome inhibition since they have a lower risk of generating drug-drug or drug-herb interactions.\n\nThe potential compounds that were found were; ZINC01568476, ZINC01568479, ZINC96330915, ZINC96330916, ZINC01568477, ZINC01568478, ZINC05131962, ZINC44401823, ZINC00351669, ZINC19364232, ZINC19364558, ZINC23477159, ZINC22947967 and ZINC22947968 as shown in Table 1. These are compounds that had better binding energies compared to the standard drug, lacked CYP inhibition, lacked substrate for P-glycoprotein, and were in toxicity class V or VI.\n\nAdditional in vivo and in vitro research should be carried out on the following potential compounds; ZINC01568476, ZINC01568479, ZINC96330915, ZINC96330916, ZINC01568477, ZINC01568478, ZINC05131962, ZINC44401823, ZINC00351669, ZINC19364232, ZINC19364558, ZINC23477159, ZINC22947967, and ZINC22947968.\n\nThe analogs can also be tested for other indications where the drugs, ethambutol, streptomycin, pyrazinamide, amikacin, and cycloserine were used apart from multidrug-resistant tuberculosis.",
"appendix": "Data availability\n\nHarvard Dataverse: Identification of potential compounds for the management of multidrug-resistant tuberculosis using computational methods. https://doi.org/10.7910/DVN/NJHPC3 (Muturia, 2023).\n\nThis project contains the following underlying data:\n\n- The docking scores of the drugs and their analogs.\n\n- Pharmacokinetic profiles of the standard compounds and the analogs.\n\n- Swiss target predictions of the compounds.\n\n- Toxicity profile of the standard compounds and the analogs.\n\n- 2D visualizations of the compounds and their analogs in iscovery studio.\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAvogadro: an open-source molecular builder and visualization tool. Version 1. XX.n.d.Reference Source\n\nBanegas-Luna AK, Cerón-Carrasco AJ, Pérez-Sánchez H: A review of ligand-based virtual screening web tools and screening algorithms in large molecular databases in the age of big data. Future Med. Chem. 2018; 10(22): 2641â2658. PubMed Abstract | Publisher Full Text\n\nCentre of Disease Control and Prevention: Tuberculosis.2016. Reference Source\n\nDaina A, Michielin O, Zoete V: SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness, and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017; 7: 42717. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGadaleta D, VukoviÄ K, Toma C, et al.: SAR and QSAR modeling of a large collection of LD50 rat acute oral toxicity data. J. Cheminform. 2019; 11: 58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoodman & Gilman: The Pharmacology Basis of Therapeutics. 13th ed.McGraw Hill; 2018.\n\nIsmail N, Ismail F, Omar SV, et al.: Drug resistant tuberculosis in Africa: Current status, gaps and opportunities. Afr. J. Lab. Med. 2018; 7(2): 781. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuturia W: IDENTIFICATION OF POTENTIAL COMPOUNDS FOR THE MANAGEMENT OF MULTIDRUG-RESISTANT TUBERCULOSIS USING COMPUTATIONAL METHODS. [Dataset]. 2023. Harvard Dataverse, V1, UNF:6:UdaIbORIS25nuuZpGamogw== [fileUNF]. Publisher Full Text\n\nMusa BM, Adamu AL, Galadanci NA, et al.: Trends in prevalence of multi-drug resistant tuberculosis in sub-Saharan Africa: A systematic review and meta-analysis. PLoS One. 2017; 12(9): e0185105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNational Center for Biotechnology Information: PubChem Compound Summary for CID 37768, Amikacin.2022a. Retrieved September 29, 2022. Reference Source\n\nNational Center for Biotechnology Information: PubChem Compound Summary for CID 1046, Pyrazinamide.2022b. Retrieved September 29, 2022. Reference Source\n\nNeves BJ, Braga RC, Melo-Filho CC, et al.: QSAR-Based Virtual Screening: Advances and Applications in Drug Discovery. Front. Pharmacol. 2018; 9: 1275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOgari CO, Nyamache AK, Nonoh J, et al.: Prevalence and detection of drug-resistant mutations in Mycobacterium tuberculosis among drug naïve patients in Nairobi, Kenya. BMC Infect. Dis. 2019; 19: 279. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOmbura IP, Onyango N, Odera S, et al.: Prevalence of Drug Resistance Mycobacterium Tuberculosis among Patients Seen in Coast Provincial General Hospital, Mombasa, Kenya. PLoS One. 2016; 11(10): e0163994. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeung KJ, Keshavjee S, Rich ML: Multidrug-Resistant Tuberculosis and Extensively Drug-Resistant Tuberculosis. Cold Spring Harb. Perspect. Med. 2015; 5(9): a017863. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWishart DS, Feunang YD, Guo AC, et al.: DrugBank 5.0: a major update to the DrugBank database for 2018. Nucleic Acids Res. 2018; 46(D1): D1074âD1082. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: A report about antimicrobial resistance.2021."
}
|
[
{
"id": "208875",
"date": "05 Oct 2023",
"name": "Manish Dwivedi",
"expertise": [
"Reviewer Expertise Membrane protein biochemistry",
"drug discovery",
"Immunoinformatics",
"Biophysics."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have performed the basic protocol for proposing the compounds as drug lead against. These experimentations need more data to support the conclusion.\n\nThe MD simulation step is missing which is very much required to propose compounds as a possible drug candidate. I strongly suggest authors to perform MD simulation to draw the final conclusion. Moreover, docking energies should be presented for all the docking compounds in tabulated form. It would assist readers to compare the parameters with the standard drugs.\nThe current form of the manuscript needs major revision before indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "208877",
"date": "23 Oct 2023",
"name": "Grace Mugumbate",
"expertise": [
"Reviewer Expertise Computer-aided drug discovery",
"Medicinal Chemistry"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented a manuscript with the title âIdentification of potential compounds for the management of multidrug-resistant tuberculosis using computational methodsâ, that covers a very important study area. Multidrug resistant TB causes hundreds of deaths annually, therefore there is need to continually identify new anti-tuberculosis compounds. However, there is need to improve the work by addressing a number of concerns.\nThe work lacks detail and depth, the introduction need to be revised so that it clearly highlights the background, current status including statistics to support the arguments, the challenges and objectives of the study.\n\nAlthough the authors had an idea of was supposed to be done, the study design is not clear and technically sound. The techniques used in the study are very powerful tools used in drug discovery to identify new bioactive compounds (hits). However, more thorough description of the design is require.\n\nThe protocol should be clearly stated to allow for replication, and all techniques used need to be explained in detail. For example how the data was extracted and curated was not stated. The docking protocol lacked detail, the general procedure involves protein/target preparation, ligand preparation, validation of the protocol, the production stage, data collection and analysis including statistical analysis which should be described in detail. The description should be specific and clearly stating the protein structures used, and the grid and docking parameters used. The lack of detail in the methods makes the work difficult to replicate. Presentation of the results should include clear protein â ligand interaction figures. Minimising the docked complexes should eliminate unfavourable contacts. The structures of some of the compounds must be corrected. Its unfortunate that the results were not thoroughly discussed. The Authors are encouraged to read as many published articles as possible for them to improve the work.\n\nNo indication of statistical analysis was added, this is important in data analysis.\n\nThe Authors are commended for providing a link to the source data.\n\nThe Authors attempted to draw up a conclusion based on the presented data, however lack of clearly and adequately presented results had an impact on the quality conclusions.\nIn its current state this article to be suitable for indexing. The Authors are encouraged to seriously consider the comments presented for them to improve the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-298
|
https://f1000research.com/articles/12-295/v1
|
17 Mar 23
|
{
"type": "Research Article",
"title": "Queervertising: An empowerment tool for the gay men and lesbian community",
"authors": [
"Patricia P. Iglesias-Sánchez",
"Carmen Jambrino-Maldonado",
"Carlos de las Heras-Pedrosa",
"Rafael DÃaz-Tendero",
"Patricia P. Iglesias-Sánchez",
"Carlos de las Heras-Pedrosa",
"Rafael DÃaz-Tendero"
],
"abstract": "Background: Todayâs society clams to be more inclusive, but there has been a lack of practical examination of this area. This study analyses how advertising and society interact and evolve in parallel, with advertising seeking to balance more traditional representations â in accordance with the Mirror Theory â with mainstreaming, which can influence social change. In this case, analysis is focused on the homosexual community. Methods: A content analysis of audiovisual advertising in Spain from the 1960s to 2021 is carried out in addition to a review of historical milestones and legislation. Results: The results evidence the transformation of advertising. The main findings show a shift from the total invisibility of the gay men and lesbian community in the 1960s to effective and respectful integration today. Conclusions: Queervertising is proposed as a new theoretical concept as the result of gender and sexual diversity being identified in advertising over time. The inclusion of gay men and lesbians in advertising is a current trend that, moreover, offers a challenge for brands. Although this turnaround in advertising creativity should be highlighted and recognized as being to some extent responsible for changes and social evolution, the commercial messages which are found today are still not always disruptive or excessively explicit, in order to avoid some rejection by audiences.",
"keywords": [
"Queer",
"Advertising",
"Gay men and lesbiansâ community",
"LGBTIQ+",
"Brands",
"gender identity",
"GLBTQ",
"LGBT"
],
"content": "Introduction\n\nMinorities have captured the attention of researchers from different disciplines although the societal impact from their representations in advertising remains a current question under exploration (Branchik & OâLeary, 2016; McDonald et al., 2020; Taylor & Costello, 2017; Yu, 2018; Zmuda, 2014). The dynamic interaction between society and advertising was pointed out by Pollay (1986, p. 18): âAdvertising is without a doubt a formative influence in our cultureâ, and continues to be supported by recent research, such as by Akestam et al. (2017) or McDonald et al. (2020), who assert that advertising reflects the times as well as social norms. In the same way that values change, advertising adapts and connects with the society on which it depends (Shinoda et al., 2021). In particular, the treatment of women in advertising and the representation of stereotypes and roles, as well as womenâs evolution and ability to contribute to their empowerment, has been extensively explored (Grau & Zotos, 2016), even introducing the concept of femvertising by Bahadur (2014). By extension, racial minorities have been addressed (Zmuda, 2014) and more recently the inclusion of gay men and lesbians in advertising imagery (Hetsroni, 2011; Holz Ivory, 2019; Iribure Rodrigues & Gallina Zanin, 2014; Madinga et al., 2020; McDonald et al., 2020; Oakenfull et al., 2008).\n\nIt is precisely the gay men and lesbian community that inspires the development of this research work. This group has also traditionally been stigmatized or made invisible in advertisements (McDonald et al., 2020; Ragusa, 2005). The purpose of this research is to delve deeper into how advertising and society interact with respect to the inclusion and positive treatment of gay men and lesbians, along with exploring the development of the group itself and the progressive recognition of their rights.\n\nTo achieve this objective, a content analysis of audiovisual advertising from 1960 to 2021 in Spain is used. Not only is representation quantified, but also, images and messages regarding sexual and amorous diversity are analyzed.\n\nThis study suggests that advertising simultaneously reflects the society of the moment while contributing to its evolution by promoting relevant social changes: tolerance, social inclusion, equality, womenÂŽs empowerment. In this case, the focus is normalization and inclusion of the LGBT community. Beyond advertising that has a cause, it is assumed that advertisersâ interest in connecting with the gay men and lesbian community and the ethics of the brands themselves results in the use of a broader and more inclusive imagery. Consequently, this âQueervertisingâ that takes place gives rise to a new theoretical concept, to analyse the phenomenon, and it provides a new insight into the core of advertising and social changes for researchers. This means reframing the research of advertising and including gay men and lesbians and contributing with practical ideas about the open-mindedness of brands as they seek to engage with stakeholders. There are identifiable themes of firstly, connecting with the gay and lesbian community and secondly, aligning with the increasingly gay-friendly attitude of society. Overall, Queervertising as a concept extends complexity and is complementary to the implementation of two seemingly opposing approaches by brands. On the one hand, their approach involves reaching out and empathizing with a traditionally marginalized group (LGBTIQ+s) and, on the other hand, searching for a balance because if the group portrayal in ads contradicts some consumer beliefs, the experience can be uncomfortable (Hetsroni, 2011; Ruggs et al., 2018). This theoretical proposal finds its parallel in femvertising (Bahadur, 2014) and acknowledges advertisingâs ability to empower, support and promote sexual and amorous diversity in a way that is respectful and consistent with the current reality.\n\nThis paper is structured as follows. After this introduction, a review of the literature on gay men and lesbian imagery in advertising is conducted. The section then explains the methods of this study. The main results are presented next, followed by a discussion. The conclusions of the work then follow. Finally, the main limitations of the study, its practical implications, and future lines of research are considered.\n\n\nTheoretical framework\n\nA review of the literature shows that the most prolific time-frame with regard to the topic of gay men and lesbians and advertising has occurred in the last 10 years, coinciding with greater acceptance and the progressive disappearance of associated stigmas (Branchik & OâLeary, 2016). From 2000 onwards, the visibility of gay men and lesbians in advertising has been increasing (Branchik & OâLeary, 2016; Iribure Rodrigues & Gallina Zanin, 2014; McDonald et al., 2020). From this prism, a dual directionality and interaction of society and advertising is assumed, whereby advertising preserves traditional and majority values, as well as becoming a mainstreaming tool that favours social advances (Ã
kestam et al., 2017; McDonald et al., 2020; Mikkonen, 2010). In this framework, it is necessary to introduce the premises of the Mirror Theory (Abrams, 1953; Holbrook, 1987), which defends the idea that advertising reproduces the same patterns, behaviours and customs of the society in which it operates. Likewise, advertising can exercise its social function by bringing about social evolution and changes in favour of special collectives (Bahadur, 2014; Feng & Wu, 2009; Fowler & Thomas, 2015; McDonald et al., 2020; Zmuda, 2014). Regarding the focus of this research work, according to Schmidt and Stocker (2013, p. 180): âadvertising and films evoke the masculine and feminine universe â simultaneously they introduce changes according to the changes experienced in societyâ. Therefore, advertising represents both the most deeply rooted social standards and those new trends or more progressive scenarios, even favouring their advancement.\n\nFor brands, the positive effect of advertising is undisputed and it is often used to strengthen a connection with the target demographic. Ã
kestam et al. (2017) empirically demonstrate that those ad campaigns that include respectful representation of the LGBTIQ+ community increase the empathy of the recipients towards the company and the rest of society. Beyond the reputational benefit, Cunningham and Melton (2014) conclude that incorporating this theme in the creative axes results in improved business performance. Taylor and Costello (2017) indicate that in addition to improving performance, it contributes to the social responsibility of the brand. However, this effect does not occur as a generalizable maxim for all companies and for all targets, as using a cause for advertising and the consequent linked imagery must be managed with caution, because it can also create a critical environment if controversial or disruptive issues are involved (Gong, 2020; Holz Ivory, 2019).\n\nThe Social Identity Theory proposed by Taylor and Costello (1985) has been widely used in research works to show that the recognition of a portrayal that involves membership of a social group enhances peopleâs self-image. Consequently, consumers can use brands to form their self-concept (Akermanidis & Venter, 2014). Several works evidence the contribution that can be made by advertising imagery to individualsâ sexual identity (Chae et al., 2016; Gong, 2020; Holz Ivory, 2019; Mikkonen, 2010; Oakenfull et al., 2008). As result of this, the use of gay men and lesbiansâ subculture symbols in advertising, that reflect gay/lesbian identity, for instance referencing clothes, language, lifestyle etc, have meaning for the community and generate empathy with them (Oakenfull et al., 2008). By contrast, controversy can occur. On the one hand, a brandâs image can be potentially harmed when a portion of the customer base is not represented or is negatively represented in ads (Gong, 2020). On the other hand, the portrayal of gay men and lesbianism in advertising imagery can annoy or inconvenience consumers, precisely because they donât see themselves as being represented, nor their beliefs, lifestyles etc (Akermanidis & Venter, 2014; Holz Ivory, 2019; Schmidt & Stocker, 2013). This dilemma for brands is often solved by mixing sexual advertisements that explicitly feature both gay men and lesbians and heterosexual models. Gong (2020, p. 916) points out that having âthe portrayal of both models within the same message ensures their effectiveness due to the engagement with particular circumstances, [and the] lifestyle and feelings of each groupâ. In the same vein, Bond and Farrell (2020) underscore that messages which are not explicit but are likely to be associated with both heterosexual or gay men and lesbians were generally still appealing for all targets.\n\nAccording to this principle, each person finds a relationship between their self-concept and the environment or situations with which they identify and, by extension, sexual orientation also plays a key role in this respect. However, there is limited empirical research about how gay-themed advertising affects heterosexual consumersâ perceptions (Holz Ivory, 2019; Madinga et al., 2020). These research works indicate that society has been increasingly accepting of gay men and lesbiansâ portrayals in advertising but there are significant peculiarities. For example, Holz Ivory (2019) in her study evidences that heterosexual men showed more negative attitudes towards gay male representation than towards heterosexual and lesbian ads. Madinga et al. (2020), for their part, add that the negative perception is higher in the case of heterosexual men than for women. In addition to sexuality, other variables inherent to viewers are analysed. Research works have highlighted that a positive or negative attitude to gay men and lesbiansâ portrayal in advertising can be explained due to age (there is more acceptance among young people) (Akermanidis & Venter, 2014; Schmidt & Stocker, 2013), the country of origin (developing countries are more receptive) (Akermanidis & Venter, 2014; Feng & Wu, 2009; Madinga et al., 2020), a country having strong traditional religious beliefs (Hetsroni, 2011), or the existence of gay-friendly attitudes (Madinga et al., 2020; Mikkonen, 2010; Orr et al., 2005).\n\nThe last reason explaining the recent concern for depicting the LGBTIQ+ community in advertising has to do with its interest to companies. On the one hand, there is so-called gay power due to the communityâs purchasing power and its being an important part of the population. On the other hand, showing sympathy towards the gay movement attracts gay-friendly groups, which brands also wish to connect to.\n\nIt is estimated that 6% of the world's population is LGBTIQ+ (Jacob, 2017). Specifically, Spain has the second-highest proportion of this group in the world (6.9%), behind only Germany. In addition, Spain holds first place as a gay-friendly country when it comes to its acceptance and defence of gay rights (Poushter & Kent, 2020). Although there are no official statistics, studies such as that of Oakenfull and Greenlee (2005) show that the gay and lesbian community represents the highest per capita purchasing power of all minority groups in the United States; they even refer to this target as the âpink dollarâ. This is a market often idealized by specialists because of its profitability for companies (Akermanidis & Venter, 2014). Thus, the representation of gay men and lesbians in advertisements aims to connect with this public (Iribure Rodrigues, 2017; Read et al., 2019) while favouring a brand image that respects gay-friendly targets, who develop a superior feeling for firms that favour the acceptance of the movement in their creative strategy (Ã
kestam et al., 2017; Orr et al., 2005).\n\nThe challenge for advertisers in Spain, given the high representation of the collective, as well as the countryâs categorization as gay-friendly, leads us to assume that in its advertising imagery, the collectiveâs representation may be significant, and interesting for deeper analysis.\n\nMost studies, in addition to being recent, focus on the effect of advertising content reflecting gay men and lesbians and its consequences for brands: their acceptance/rejection (Angelini & Bradley, 2010; Branchik & OâLeary, 2016; Descubes et al., 2018; Holz Ivory, 2019; Iribure Rodrigues, 2017; Madinga et al., 2020; Ruggs et al., 2018; Um, 2014), advertising recall (Angelini & Bradley, 2010; Orr et al., 2005) or even the impact on peopleâs purchasing intention (Akermanidis & Venter, 2014; Bond & Farrell, 2020; Holz Ivory, 2019; Orr et al., 2005; Um, 2014). In summary, most of the studies are conducted from a sociological and social perspective, but those that, like this one, focus on the power of advertising to promote a more inclusive society are scarcer (Gong, 2020; McDonald et al., 2020; Oakenfull & Greenlee, 2005; Ragusa, 2005). It is precisely this gap in the literature that underpins the research questions of this paper:\n\nRQ1. How has advertising imagery evolved with respect to gay men and lesbians since the 1960s in Spain?\n\nRQ2. To what extent do the representations of relationship models that appear accompany the most relevant milestones and legislation in this field?\n\nRQ3. What stance does advertising take on the diversity of love and sexuality in Spain?\n\n\nMethods\n\nIn order to analyze the interaction between advertising and the advances and social reality of the gay men and lesbian collective, a qualitative approach is chosen, specifically a content analysis of audiovisual advertising spots from 1960 to 2021, in tandem with a review of the most relevant milestones and legislation of the LGBTIQ+ movement in Spain. According to Krippendorff (2004), its use is appropriate for the observation of trends; that is, with this type of analysis we will be able to highlight the changes in advertising over the last six decades.\n\nAs mentioned, Spain has the second-highest proportion of LGBTIQ+ citizens in the world and holds first place as a gay-friendly country for its acceptance and defence of gay rights. These reasons lead us to assume that in advertising imagery, the representation of this collective may be significant and interesting for deeper analysis.\n\nThe selection of advertising spots was carried out after viewing all the spots collected in the main database of the Spanish Association of Advertisers (Asociación Española de Anunciantes, AEA); in addition, two complementary databases were added to complete the last decade, that of the El Publicista journal (El Publicista, 2021) and the Sanz'Channel (Sanz, 2016) (N=1408 spots). Finally, our sample consists of 63 spots. Only spots whose message or imagery depicted people and actions representing social relations or events that implicitly or explicitly included sexual and amorous diversity were included. The research has thus tried to identify ads with all the options or relationships beyond heterosexual âconventionalityâ. It is, overall, a census sample, and consists entirely of commercial communication by brands that directly or indirectly targeted a topic. The sample comprises 63 advertisements because, during our review, this was the total number of relevant audiovisual ads from the total found for the period 1960-2021.\n\nAdditionally, it should be added that social campaigns launched by official organizations or associations in defence of LGBTIQ+ rights were excluded, since the object of the research was purely commercial advertising. There were a wide range of brands and kinds of product involved, utilizing content related to the targeted topic: insurance companies, agrifood and drinks businesses, technology and telecom operators, among others (Table 2). The time period of analysis begins in 1960, coinciding with the first broadcasts of Televisión Española and the increase in the number of households with television sets. The spots are not distributed proportionally in each of the decades overall, due to the low number of relevant advertisements among the total number of ads broadcast over the targeted period. Thus, curiously, the same number of relevant advertisements (5) were identified for each of the decades between 1960-2010. On the other hand, for the period 2010-2021, 38 advertisements reflecting sexual and amorous diversity were found. Two issues should be noted: (1) the underrepresentation of gay men and lesbians in advertising is due to the dictatorial regime that existed in Spain until 1975; and (2), not all advertisements use the family and the couple in their creative strategy. A list of all ads selected can be found in Table 2.\n\nThe research opted for was a qualitative study using a content analysis combined with a historical analysis to explore the possible relationship between the evolution of advertising with respect to the phenomenon of liberalization and inclusion of the LGBTIQ+ community, and the consequent sociocultural and legislative progress.\n\nBasic information about the product offered, company and sector was extracted for each advertisement. General aspects were analyzed (target, slogan, etc.) and finally, details about the treatment of gay and lesbian couples in the ads. Table 1 shows the analysis sheet used for classification. Except for some variables about the identification and classification of images or discourse linked to gay men and lesbians, the variables are in relation with those commonly included in works on the treatment of advertising. Typically, this type of research includes information concerning product, sector, claim, target. The work of Molina RodrÃguez-Navas et al. (2014) stands out because it laid the foundations of the method of analysis of audiovisual fiction and advertising as sources of knowledge of the past and present. Moreover, they underline the necessity to adapt content analysis to the particular focus of adverts under consideration. For its part, the identification of stereotypes in our approach is mainly supported by variables drawn from the work of Fowler and Thomas (2015) and Olivares et al. (2020), specifically variables 7 to 12 and 15 to 17. In any event, our research work involved some adaptation because previous literature focusing on the gay men and lesbian community is really scarce. Consequently, some original variables have been introduced, with the inclusion of variables 13, 14 and 18 in our analysis. The field of study cannot be analyzed without exploring some information about types of couple, and the traditionality of family and love models. The final analysis sheet was the result of a thorough review of literature and a discussion by three researchers in the first stage. After this, the fourth researcher did an analysis test with 5 ads, randomly chosen. The overall process allowed us to refine the analysis sheet for the goals of this research work. The detailed information for each variable used has been included in the calculation sheet shown in Table 1. Results for each advert under examination were recorded using Microsoft Excel spreadsheets in order to proceed to content analysis using the software Keycoder and Cloudwords, and to enable the calculation of some details such as percentages.\n\n1 Variables 10-12 have three categories. The difference between the first and the last option is that the first one will occur mainly in advertisements from the first decades, where homosexuality was a taboo subject. In the latter, homosexuality is presented naturally, without stereotyping.\n\nTable 2 shows the list of advertisements analyzed in this research, including the name of the advertised brand, the decade of broadcast and the link to view the spot. Two researchers independently viewed the advertising directories and collections of advertisements described above to create the selection of 63 ads, and avoid bias. In cases where they did not agree on the choice of an advertisement, a third researcher made the decision on its suitability. However, this situation happened for two ads only.\n\n\nResults\n\nIn order to give a clearer structure to the results section, evidence extracted from the content analysis associated with each decade is presented. Firstly, Table 3 shows the most relevant historical and legislative facts.\n\n\n\n⢠(1936-1954) Article 431 of the Penal Code (referring to public scandal, and using âgood mannersâ as a reference) applied, so there was a subjective interpretation of the law: there was or was not an offence depending on how scandalous the observer considered it to be. However, its treatment did not vary much from how it had been treated since the end of the 19th century.\n\n⢠1936: Official Francoist condemnation of the poet and playwright Federico GarcÃa Lorca.\n\n⢠(1954-1970) In 1954, Francoism approved a reform of the 1933 Law of Vagrants and Miscreants and began to explicitly criminalize homosexuality. A change was made to article 2.2, making it possible to declare \"homosexuals, ruffians and pimps\" to be \"in a dangerous state\" and to be subjected \"to the security measures\" of the law (BOE of 17 July 1954). This consideration of homosexuals as dangerous subjects led to an increase in the number of arrests, almost all of them men.\n\n⢠On 28 June 1969, following the police raid on the Stonewall Inn LGBT pub in New York and the revolt of some customers refusing to be arrested, the Pride movement was born (Snodgrass, 2019). From that moment on, Gay Pride Day has been celebrated internationally on that day in commemoration of this event. In Spain, due to the regime, the movement did not begin until later, but this historic milestone had an international influence on the fight for LGBT rights worldwide.\n\n\n\n⢠1970-1975 - Final stage of Francoism.\n\nDÃaz (2019) The regulation of homosexuality evolved in the opposite direction to other transgressions: while it is considered that the Francoism of the 1970s began to become more flexible, in this area political control intensified, being one of the most condemned practices that did not enjoy a regulatory relaxation. Furthermore, and although the law does not explicitly allude to it, practice showed that it was not the sexual act itself that was punished, but men who broke gender roles and did not comply with the sacrament of marriage.\n\nIn 1970 there was the new Social Danger and Sexual Rehabilitation Act, which was more paternalistic in nature: homosexuals were no longer punished, but those who \"carry out homosexual practices\", and the law was not intended to be punitive but re-educational (Mora-Gaspar, 2019).\n\n⢠1973: The American Psychiatric Association eliminated homosexuality as a mental/psychological disorder by removing it from the Diagnostic and Statistical Manual of Mental Disorders, although this important milestone did not have a direct effect in Spain until the beginning of the 1990s.\n\n⢠Death of Franco, 1975.\n\n⢠1978, adoption of Law 77/1978 of 26 December 1978, amending the Law on Dangerousness and Social Rehabilitation and its Regulations, when homosexuality was directly decriminalized.\n\nSome cases were still indirectly punished under the premise of \"public scandal\". This decriminalization, however, did not initially lead to greater social acceptance: it was felt that being LGBT should remain a private matter.\n\n⢠1975-1980 â Beginning of democracy. Beginning of the social, cultural and political movement known as the Movida. Aparicio Cillán and Cimadevila Niño (2019, 17): \"Sex ceased to be taboo and many young people began to enjoy it naturally and without fear. Homosexuality and bisexuality became sexual orientations free of 'sin' and prison\".\n\n⢠Influence of the Movement on art, especially representative in the cinema. Two significant films were released in 1978: El Diputado by Eloy de la Iglesia and Un hombre llamado Flor de Otoño by Pedro Olea.\n\n\n\n⢠Influence of the Movement on art. In music: songs of protest and with social claim such as Ni tú ni nadie (1984) by Alaska and A quién le importa (1986) by Alaska y Dinarama. The censorship barrier began to be overcome and homosexual protagonists began to be made visible.\n\n\n\n⢠In the scientific field, the depathologization of same-sex attraction by the World Health Organisation took place on 17 May 1990. The idea that homosexuality and bisexuality are a mental disorder was eliminated. This day marks the International Day Against Homophobia, Transphobia and Biphobia.\n\n⢠In 1992, the Lesbian and Gay Collective of Cordoba was formed and became known by the acronym COLEGA.\n\n\n\n⢠2005: approval of same-sex marriage by 187 votes in favour, 147 against and 4 abstentions. Law 13/2005 of 1 July 2005, which amends the Civil Code on the right to marry (BOE of 2 July 2005), puts homosexuals and heterosexuals on an equal footing in terms of marriage rights, including the right to adoption.\n\n⢠The LGBT community and part of the population that accepts the normalization of this minority is rejoicing and celebrating at the same time that there are massive demonstrations called by conservative organisations against these social and legislative achievements for the gay and lesbian community.\n\n⢠In October 2006, the first monument dedicated to homosexuals in Spain was inaugurated in Sitges, an inverted pink triangle placed on the seafront. On 16 May 2009, the first monument dedicated to remembering the persecution of homosexuals during Franco's regime was inaugurated in Durango.\n\n\n\n⢠In spite of the fact some people continued to oppose the evolution of an inclusive society in terms of the sexual freedom of the LGBT collective, the law 13/2005 was appealed against in the Constitutional Court. However, the Court dismissed the case of unconstitutionality filed in 2012 (Ruling 198/2012, 6 November 2012, published in the BOE, 2012). On 20 March 2011, a memorial âin memory of gays, lesbians and transgender people who have suffered persecution and repression throughout historyâ is inaugurated in Barcelona, located in the Parc de la Ciutadella.\n\n⢠The open recognition of sexual status as a reason for non-discrimination in the public sphere â political, artistic, etc. â is taking place. On 16 December 2018, Ãngela Ponce becomes the first transgender woman to compete in the Miss Universe competition.\n\nThe variables included in the content analysis lead us to present the evolution of advertising and the treatment of gay men and lesbians within it.\n\n1960-1969: Franco's dictatorship\n\nGay men and lesbians are made invisible, being a taboo subject. In none of the advertisements viewed is there any allusion to the diversity of sexual orientations. The family model shown is univocal and traditional, with the exclusive representation of heterosexual couples. To a greater extent, young couples are shown, and depending on the product advertised, they are shown with or without children. Gender roles are usually shown in a differentiated way, which is congruent if we take into account the historical and cultural context: Francoism.\n\nAll the advertisements that were broadcast went through Franco's censorship, and no prejudices or stereotypes were broadcast through advertising. Therefore, in this decade there is a relationship between advertising treatment and the legislative and social treatment of gay men and lesbians, making them completely invisible.\n\n1970-1979: End of the dictatorship and early years of democracy in Spain\n\nAdvertisements continue to perpetuate a great difference between the gender roles of men and women, either through their content or by the way in which their storyline is delivered. For example, the mother continues to be assigned the responsibility of childcare, and the father the ownership of objects such as cars (as seen in the Nesquik ad, No. 6). In addition, it is emphasized that products such as alcohol are consumed exclusively by men (as in the case of González Byass's Soberano Cognac, with the slogan âIt's a man's thingâ, ads Nos. 9 and 10). Particularly striking is the second González Byass ad (No. 10), which even shows a scene normalizing gender violence.\n\nIn this context, an univocal and traditional family and couple model is shown. Even in advertisements that claim to be inclusive, such as Coca Cola's (No. 8), the diversity of sexual orientations is ignored. Therefore, there is still a relationship between advertising treatment and the legislative and social treatment of gay men and lesbians.\n\n1980-1989: Movements and social changes\n\nAdvertising in the 1980s experienced a process of modernization, in line with the rest of Spanish society.\n\nAs far as gender roles are concerned, there are ads in which a clear differentiation is evident (No. 11, by Monky; and No. 15, by Johnson), along with others in which they appear practically undifferentiated (No. 13, by Riera-Marsá; and No. 14, by Princesa). In the latter, a greater co-responsibility of men in domestic tasks can be observed compared to previous decades (No. 13). However, it is true that sometimes these men are still presented with a humorous and ridiculing tone, as if they are facing impossible tasks to perform (this is the case with No. 12, Scotch Brite).\n\nIn addition to all of the above, there is a greater normalization of sexuality and eroticism. For this purpose, puns and double meanings are used (advertisement No. 14, from Galletas Princesa). However, for the time being, the family model seems to remain unique and the couples represented are heterosexual and young.\n\nWe should remember that, in the 1980s, the process of protesting to demand rights and equality between heterosexuals and gay men and lesbians continued. The World Health Organization still considered gay men and lesbians âsickâ; therefore, there was still no effective integration into society. In other words, there was still a relationship between the legal and sociocultural context and the reality reflected in advertisements.\n\n1990-1999: Social openness and the evolution of equality\n\nIt should be noted that, as far as sexuality is concerned, the path of openness started in the previous decade continues. Usually, double meanings are still used when referring to this type of topic (No. 16, from Starlux; and No. 19, from Rumba Total), and advertisers manage to connect these ambiguities with the promotion of the product (for example, a woman with her partner saying âthis one does it the way I like itâ, to promote the Starlux granulated broth).\n\nDuring this decade, equality between gender roles began to be reflected more effectively. Men appear more frequently as jointly responsible for household chores and childcare (advertisements Nos. 16 and 18).\n\nIn addition, announcements begin to be made that little by little highlight the uniqueness and independence of being women. Ad No. 20, from Woman magazine, is a good example. Although this modernization was rather lax, it opened the door for women to want to feel beautiful in themselves, not for their husbands (the attitude changes regarding submission, from advertisements such as No. 1) and, the most interesting thing in our field study, opened the door to women who do not like men.\n\nIn the same way, it is worth noting the clear contrast in children's social relations between the advertisements of the 1960s/1970s and those made in the 1990s: in the former, boys and girls are shown playing separately, in different groups and with different toys (spots No. 2 and No.7), while in the latter they are shown playing together and cooperating (adverts Nos. 3 and 17).\n\nThe evolution in the concept of family and love follows the same lines as the evolution in gender roles: a lax openness. A representative case is No. 18, in which the Renault Laguna automobile is promoted and a father appears as the sole caregiver for his son, without a partner; therefore, it could favour selective perception by the viewer to consider him as a gay man, heterosexual or bisexual. However, given the social context in which it is inserted, the advertiser's intention was probably simply to awaken the patriarchal protective instinct.\n\nFinally, the ad for the compilation album Rumba Total deserves a special mention. Although advertising about the transsexual collective is not the subject of this research, ad No. 19 is probably one of the first to allude to the LGBTIQ+ community in a clear way. In it, Cristina âLa Venenoâ, a popular transgender woman of the 1990s, is presented as the protagonist, and a play on words is made between the pack of songs offered and her genitalia, recreating the famous leg-crossing scene from the movie Basic Instinct.\n\nTaking into account the above and that during this decade the depathologization of gay men and lesbianism took place, we can conclude that during the decade there was also a relationship between social advances and advertising treatment.\n\n2000-2009: Normalization and acceptance at the social and legislative levels\n\nWith the arrival of the new millennium, the normalization of gender and sexual diversity in advertising is consolidated. For example, sexuality and nudity began to be shown more explicitly, without the need to use ambiguities as in previous decades. The approval of same-sex marriage coincided with a greater normalization, in advertising terms, of the imagery related to the collective.\n\nAs far as gender roles are concerned, the co-responsibility of household chores is fully achieved as far as their representation in advertising is concerned, going beyond the âaesthetic chores on cameraâ (cooking and taking care of the children, basically): one example, among others, is Bosch ad No. 22, which shows a man doing the ironing. In addition, equality is starting to move more in the direction of femvertising. Nevertheless, ads such as Heineken's (No. 24) continue to depict differences by gender: girls are interested in clothes and boys interested in beer.\n\nThese are turbulent times for reflecting family diversity in advertising. In a very clear way, there coexists an advertising that reflects the most traditional society and another âmore transgressiveâ one that represents the social evolution regarding the gay men and lesbian collective. Thus, we find advertisements that, even though they are clearly endeavouring to reflect the variety of couples in love, as they see it, do not show gay and lesbian couples (advertisement No. 23, IKEA). Others directly sexualize relationships between women and objectify them (ad No. 21, Musicón del Verano). In the most extreme case, we find companies such as the communication group Intereconomia (No. 25) that punish the demands of the rights of the collective, using an advertising style that tries to instill âfearâ (Figure 1). That is to say, some advertisements are responsible for reflecting the discontent generated in part of the population by the approval of same-sex marriage.\n\n(Original source: Las Malas Lenguas (Intereconomia TV, 2009).) Translated text from the ad: Do you want this to be an example for your children?.\n\nHowever, the majority do support an inclusive and diverse society. Therefore, in this instance, according to the spots in our sample there is not a complete relationship between advertisingâs treatment of the gay men and lesbian community and the legislative framework.\n\n2010-2021: Current decade\n\nThe period 2010-2021 sees a notable increase in the number of appearances of gay men and lesbian couples and individuals in audiovisual advertising. However, although the growth is considerable compared to previous decades, the percentage with respect to the total number of advertisements (of a commercial nature) is still not very significant.\n\nBased on the content analysis, Table 4 shows the congruence between historical contexts and advertising related to the gay men and lesbian community.\n\nFirst, it is worth noting the wide variety of products that include depictions of the gay men and lesbian collective. We find everything from dating apps to soft drinks and cleaning products; naturally, this diversity is reflected in the wide range of sectors present in the sample (Figure 2). The sector that includes the most depictions of the gay men and lesbian community is beauty and hygiene (23.7%), followed by food and entertainment (18.42% each).\n\nSource: Own elaboration.\n\nThe research on slogans shows that reference is made to the gay men and lesbian collective depending on the tone of the advertisement. That is to say, if it has an educational or vindicatory function (as in the case of Burger King's ad No. 50 and La Sexta TV channel's ad No. 43), words that aim to empower the collective, such as âprideâ, are usually included. The use of this type of slogan tends to have a seasonal character, being especially frequent during the LGBTIQ+ Pride celebration (end of June). However, there is another group of ads that simply integrate and normalize gay men and lesbian protagonists within the story told in the ad (for example, No. 28), so the slogan is usually related to the product (Figure 3).\n\nSource: Own elaboration.\n\nRegarding the relationship between the representations of the gay men and lesbian community in the advertising spots and the fact that both were the target audience of the brands, we observed that gay men and lesbian couples and individuals are reflected in advertising regardless of whether or not they are the target. Specifically, only in 2 of the 38 cases that show situations or environments related to the collective, is sexuality a relevant segmentation criterion (spots Nos. 49 and 53). The rest are products aimed at the general public, of which gay men and lesbians are only a part.\n\nOn the other hand, the variable âexplicitness of the messageâ, that is, the way in which the reference to the gay men and lesbian community is produced (whether directly or through ambiguities and double meanings) indicates that 71% of the ads in the sample do so explicitly. The words and expressions exchanged by the partners (dialogues showing mutual affection, use of romantic appellatives such as âsweetheartâ, etc.) as well as the images (the way they look at each other, hug and kiss, for example) are the instrument for this. The remaining 29% of ads show the relationship between the protagonists in an ambiguous way. The main motivation to provoke this ambiguity is probably to favour a more selective perception, i.e. that the viewer is the one who assigns the label of âfriendsâ or âcoupleâ according to his or her reality. This issue leads us to evidence that the relationship between the characters is often ambiguous (No. 27, No. 48 and No. 54).\n\nAs for stereotypes, it is worth remembering that the three ways of expression are the storyline way (reproducing clichés about gay men and lesbians, such as promiscuity), the visual way (reproducing clichés about their clothing, appearance, gestures, etc) and the sound way (stereotypes about the way they speak and express themselves). The results show that we have gone from an almost total invisibilization in previous decades, to an inclusion of the collective made from respect, and with the absence of these clichés. Thus, the ads from this decade reflect a diverse family model. However, there is a discriminatory advertisement (spot No. 61), but it is an isolated and specific case (Figure 4), therefore, the gay men and lesbian community as a sexual and amorous option ends up becoming visible and normalized.\n\nTranslated text from the ad: Image 2: Gay Pride Day; Image 4: 364 days of Pride for ordinary people.\n\nRegarding the types of couples represented, it is worth mentioning that we find a great diversity of ways of showing them: while in some spots only one typology is shown (for example, a gay couple), in others the range is wider and more diversity is shown (for example, gay men and lesbians and heterosexuals being co-protagonists). Specifically, in 47.37% of the ads, gay men and lesbian couples share space with heterosexual couples (Figure 5).\n\nSource: Own elaboration.\n\nIn the same line, the distribution of percentages of the representation of traditional family models in the decades studied gives a concrete view on the evolution of openness to amorous diversity in the last targeted period (2010-2021). The traditional family and couple model is represented in 68% of ads while the diverse family and couple models appear only in 16% of ads in the years from 1960 to 2009. It should be emphasized that 16% of advertisements without a clear model are included. By contrast, the most contemporary era (2010-2021) evokes diverse family and couple models in 100% of cases.\n\nAnother noteworthy variable where there are contrasting data between decades is gender roles. First, the roles are well differentiated (totally or sufficiently) in earlier ads while in recent years, advertisers opt for portraying no differences in most of the cases (Table 5).\n\n1 Gender roles were measured with a Likert scale with 5 points. No. 5, âTotally differentiatedâ, means the gender roles of characters in ads follow the traditional pattern of women and men in society.\n\nIt can be determined that in the last decade analyzed, advertising is mostly related to social and legal changes in Spain in terms of affectionate relationships. Thus, a respectful inclusion of the gay men and lesbians collective is made, trying to reflect an increasingly common social reality, so that brands can address any type of consumer regardless of their sexual orientation.\n\n\nDiscussion\n\nThis research work provides new insights about how advertising and society interplay and feed each other. Although this assertion was already established by Pollay (1986) as well as in more recent works such as those by Schmidt and Stocker (2013) and McDonald et al. (2020), the focus on sexual and amorous diversity evidences how companies continuously seek a balance in their advertising between the reflection of what society is, with its stereotypes and strongest beliefs, and the changes/advances that are inevitably generated within it.\n\nThe analysis of advertising content and the historical review leads us to evidence the close relationship of society, legislation and advertising. This struggle in the creative strategy responds to the need to connect with the target audience, being cautious with the possible reticence and rejection of a part of it but being consistent with representation in its imagery of minorities, who interestingly commercially crave to be reflected. In this regard, the findings coincide to some extent with the research works of Akermanidis and Venter (2014), Hetsroni (2011), Chae et al. (2016), Akestam et al., (2017), Iribure Rodrigues (2017), Holz Ivory (2019), Read et al. (2019), Madinga et al. (2020) and Cheah et al. (2021). At the same time, it coincides with what is indicated by Taylor and Costello (2017) â advertising that includes through images or messages the targeted topics is designed to express awareness and the corporate social responsibility of companies.\n\nThe analysis of advertising in Spain over six decades, since the beginning of television in Spain, shows a progressive increase in the representation of the gay and lesbian collective, as do other investigations where a historical review of the presence of this group in advertising is undertaken (Branchik & OâLeary, 2016; Descubes et al., 2018; Iribure Rodrigues & Gallina Zanin, 2014; McDonald et al., 2020; Ragusa, 2005). Nevertheless, the percentage of ads in which the gay men and lesbian imagery is explicitly or implicitly present remains proportionally low over the total volume of television spots analyzed.\n\nThe research evidence shows how it is common to use double meanings or the use of messages and/or images that can be interpreted openly to connect with one or another audience, as pointed out by the Social Identity Theory proposed by Taylor and Costello (1985). This common behaviour by the brands analyzed in recent decades coincides with Read et al. (2019), Ruggs et al. (2018) and Gong (2020), who emphasize the existence of what they call âmixed sexual adsâ, i.e., spots in which the representation of gay men and lesbian and heterosexual themes is ambiguous, undertaken to address some without risking the arousal of sensitivities if there is a section of the viewers reluctant to accept the former.\n\nAttention has been drawn to an issue worthy of more detailed reflection in future research. Coinciding with previous research, such as that of Ragusa (2005), there is greater coverage of gay men than of lesbians in the advertising imagery. This is a fact that contrasts with the findings of studies that conclude that the heterosexual population shows greater acceptance/less criticism of images depicting relationships between two people of the same gender if they are women than if they are men (Descubes et al., 2018; Madinga et al., 2020).\n\nBased on the above, the research questions can be answered by showing that there has been a positive change, consistent with the times, in the advertising imagery and its treatment of gay men and lesbians since the 1960s in Spain (RQ1). On the other hand, the representations seem to have a significant relationship with the major milestones and legislation in this field (RQ2), although it can be seen that this evolution is taking place cautiously and coexisting with more classical representations. Finally, love and sexual diversity is more openly expressed, but with a great deal of recourse to interpretable messages or images in order to connect with one or another target according to their identity. Thus, the stance adopted in advertising continues to balance between the disruptive and the classic (RQ3).\n\nThe research goes a step further by showing that advertising and the times are moving forward hand in hand, favouring a cautious change that ends up adapting to the most disruptive demands of a part of society. To this end, stigmas, stereotypes or clichés associated with gay men and lesbians are being blurred and inclusive advertising is being projected. In the most recent years of the analysis, discriminatory messages are hardly detected, curiously in a lower proportion for Spain than in the work of McDonald et al. (2020), focused on American advertising. The research considers the visualization itself in adverts as a step forward that once again emphasizes the social function of advertising. Consequently, the contribution of this work lies not only in its impact on the analysis of advertising imagery, since it also introduces a theoretical contribution that finds its parallel in the term femvertising used by Bahadur (2014): Queervertising. This concept underlines the adaptation of brands to the social, political and economic changes of the LGBTIQ+ community, as well as their contribution to its empowerment, visualization and inclusive recognition in society.\n\n\nConclusions\n\nAdvertising reflects society, as well as supports new movements or relevant changes that are forged within it. In this sense, the contribution of the concepts of gender and sexual diversity in advertising represents a new prism through which to advance in studies on the social function of advertising, and on being inclusive for historically invisible and stigmatized minority groups. Advertising thus becomes a mainstreaming and empowerment tool for this minority. Sexual and amorous diversity is progressively normalized in advertising creative axes, although the volume of these representations is not so significant in the total number of minutes of television advertising achieved.\n\nLikewise, the effort of companies to include messages that connect with gay men and lesbians in their advertising, but without being excessively explicit or transgressive in order to avoid possible rejection by part of the audience reluctant to accept gay men and lesbian imagery, is underlined. The interaction between society, through the review of key milestones and Spanish legislation for this collective, and advertising seems to show a significant relationship. Based on the above, it can be affirmed that advertising favours changes and social evolution through its commercial messages, both as a means of connecting with the LGBTIQ+ community and to develop its corporate social responsibility and position itself as a gay-friendly communicative tool.\n\nThere is no doubt that in future years, Queervertising will be a fertile area in terms of research. The main limitation of this work is the difficulty in establishing a determinant cause-effect relationship between legislation, movements and social milestones linked to gay men and lesbians and their projection in advertising imagery. However, the proposed methodology has evidenced solid relationships between these dimensions and clarifies the evolution over time of the inclusion of gay men and lesbians in advertising, as well as significant changes in the treatment of this group.\n\nThis work has had to focus on gay men and lesbians without it being possible to consider other groups such as transsexuals or bisexuals due to the lack of written or audio text, symbolism or images of them in the advertising under analysis. Therefore, it is presented as a future line of research of interest to complete the vision of the phenomenon studied.\n\nLikewise, it would be useful to make an international comparison incorporating other European countries or even contrasting the results with countries with less legislative progress and recognition of the rights of the LGBTIQ+ community.\n\nDespite the usefulness of content analysis, a final future line of research would be to triangulate the data to include the perception and motivations of three main groups. Firstly, by approaching companies directly through a focus group or in-depth interviews. Likewise, it would be interesting to know how advertising copywriters take on this challenge of inclusive society in advertisements. Finally, by involving gay men and lesbians and heterosexual audiences in the fieldwork with the aim of delving into and comparing their perceptions and feelings regarding ads.\n\nCorporate advertising changes and evolves along with society. The most relevant practical implication would be to make brand owners aware of the suitability of exercising their social function in a more disruptive and less cautious way, becoming agents promoting changes and innovative perspectives in society and positively relating their brands as gay-friendly. Urging advertisers and researchers to work for a fairer and more inclusive society through their messages is a complex and ambitious, but unavoidable and necessary challenge.",
"appendix": "Data availability\n\nAs detailed, secondary data for analysis were obtained from the Spanish Association of Advertisers (Asociación Española de Anunciantes, AEA); and from complementary databases provided by the El Publicista journal (El Publicista, 2021) and the Sanz Channel (Sanz, 2016).\n\nWe have provided the data produced by our content analysis of advertisements, using the analysis sheet in Table 1, at Figshare; âData â Queervertising â reviewedâ is at: https://doi.org./10.6084/m9.figshare.21953030.v1 (Iglesias-Sánchez, 2023).\n\nThe Figshare project contains the following underlying data:\n\nâ Data-Queervertising-reviewed.xlsx\n\nData are provided under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe appreciate the commitment that advertisers and advertising creatives sometimes make to a more balanced, fair and inclusive world.\n\n\nReferences\n\nAbrams MH: The mirror and the lamp: Romantic theory and the critical tradition. Oxford University Press;1953\n\nAkermanidis E, Venter M: Erasing the line between homosexual and heterosexual advertising: A perspective from the educated youth population. The Retail and Marketing Review. 2014; 10(1): 50â64.\n\nÃ
kestam N, Rosengren S, Dahlen M: Think about it â can portrayals of homosexuality in advertising prime consumer-perceived social connectedness and empathy? Eur. J. Mark. 2017; 51(1): 82â98. Publisher Full Text\n\nAngelini JR, Bradley SD: Homosexual Imagery in Print Advertisements: Attended, Remembered, but Disliked. J. Homosex. 2010; 57(4): 485â502. Publisher Full Text\n\nBahadur N: Femvertising ads are empowering women - and making money for brands. Huffington Post;2014.Reference Source\n\nBond BJ, Farrell JR: Consumer Responses to Print Advertisements Featuring Gay Males Over Time. Sex. Cult. 2020; 24(5): 1432â1442. Publisher Full Text\n\nBranchik BJ, OâLeary B: Funny, scary, dead. J. Hist. Res. Mark. 2016; 8(4): 524â544. Publisher Full Text\n\nChae Y, Kim Y, Johnson KKP: Fashion brands and gay/lesbian-inclusive advertising in the USA. Fashion, Style & Popular Culture. 2016; 3(2): 251â267. Publisher Full Text\n\nCheah, Teah M, Lee S, et al.: Straight eye for the queer ad: Attitudes, skepticism, inferences of manipulative intent and willingness to buy. Asia Pac. J. Mark. Logist. 2021; 33(5): 1220â1238. Publisher Full Text\n\nCunningham GB, Melton N: Signals and Cues: LGBT Inclusive Advertising and Consumer Attraction. Sport Mark. Q. 2014; 23: 37â46.\n\nDescubes I, McNamara T, Bryson D: Lesbiansâ assessments of gay advertising in France: Not necessarily a case of âLa Vie en Rose?â J. Mark. Manag. 2018; 34(7â8): 639â663. Publisher Full Text\n\nEl Publicista: YouTube Channel specialised in advertising communication and marketing. El Publicista;2021.Reference Source\n\nFeng J, Wu DD: Changing ideologies and advertising discourses in China: A case study of Nanfang Daily. J. Asian Pac. Commun. 2009; 19(2): 218â238. John Benjamins. Publisher Full Text\n\nFowler K, Thomas V: A content analysis of male roles in television advertising: Do traditional roles still hold? J. Mark. Commun. 2015; 21(5): 356â371. Publisher Full Text\n\nGong ZH: Crafting Mixed Sexual Advertisements for Mainstream Media: Examining the Impact of Homosexual and Heterosexual Imagery Inclusion on Advertising Effectiveness. J. Homosex. 2020; 67(7): 916â939. Publisher Full Text\n\nGrau SL, Zotos YC: Gender stereotypes in advertising: A review of current research. Int. J. Advert. 2016; 35(5): 761â770. Publisher Full Text\n\nHetsroni A: Pluralistic media ignorance: Presence and causes. Soc. Sci. J. 2011; 48(2): 324â334. Publisher Full Text\n\nHolbrook MB: Mirror, Mirror, on the Wall, Whatâs Unfair in the Reflections on Advertising? J. Mark. 1987; 51(3): 95â103. JSTOR. Publisher Full Text\n\nHolz Ivory A: Sexual Orientation as a Peripheral Cue in Advertising: Effects of Modelsâ Sexual Orientation, Argument Strength, and Involvement on Responses to Magazine Ads. J. Homosex. 2019; 66(1): 31â59. PubMed Abstract | Publisher Full Text\n\nIglesias-Sánchez P:DATA- Queervertising-reviewed.xlsx. figshare. Dataset. 2023. Publisher Full Text\n\nIntereconomia TV: Dia del Orgullo Gay [Advertising].2009.Reference Source\n\nIribure Rodrigues A: Entre as representações e as repercussões das homossexualidades: Uma análise da publicidade veiculada na TV aberta e seus desdobramentos na rede social. Conexao - Comunicaçao e Cultura. 2017; 16(32): 135â155. Publisher Full Text\n\nIribure Rodrigues A, Gallina Zanin V: As representações das homossexualidades em anúncios veiculados na televisão brasileira entre os anos de 2008 e 2012. Conexao - Comunicaçao e Cultura. 2014; 13(25): 99â119.\n\nJacob L: Americans are twice as likely as Europeans to identify as LGBTQ. Dalia Research;2017.Reference Source\n\nKrippendorff K: Content Analysis: An Introduction to its Methodology. 2nd ed. Sage Publications, Inc.;2004.\n\nMadinga NW, Broster P, Kappatos A, et al.: Exploring heterosexual responses to lesbian and gay-themed advertisements in South Africa. Communitas. 2020; 25: 1â20. Publisher Full Text\n\nMcDonald RE, Laverie DA, Manis KT: The Interplay between Advertising and Society: An Historical Analysis. J. Macromark. 2020; 41: 585â609. Publisher Full Text\n\nMikkonen I: Negotiating subcultural authenticity through interpretation of mainstream advertising. Int. J. Advert. 2010; 29(2): 303â326. Publisher Full Text\n\nMolina RodrÃguez-Navas P, Simelio Solà N, Ibarz Gelabert J: Televisión, cine y publicidad, fuentes de conocimiento del pasado y del presente. Historia y Comunicación Social. 2014; 18: 461â471. Publisher Full Text\n\nOakenfull GK, Greenlee TB: Queer eye for a gay guy: Using market-specific symbols in advertising to attract gay consumers without alienating the mainstream. Psychol. Mark. 2005; 22(5): 421â439. Publisher Full Text\n\nOakenfull GK, Mccarthy MS, Greenlee TB: Targeting a Minority without Alienating the Majority: Advertising to Gays and Lesbians in Mainstream Media. J. Advert. Res. 2008; 48(2): 191â198. Publisher Full Text\n\nOlivares-Delgado F, Iglesias-Sánchez PP, Benlloch-Osuna MT, et al.: Resilience and Anti-Stress during COVID-19 Isolation in Spain: An Analysis through Audiovisual Spots. Int. J. Environ. Res. Public Health. 2020; 17(23). PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrr R, Van Rheede Van Oudtshoorn GP, Kotzé TG: The perceptions of consumers aged 18-30 of âlesbianâ appeals in advertising. Communicare: J. Commun. Sci. S. Afr. 2005; 21(1): 49â68.\n\nPollay RW: The Distorted Mirror: Reflections on the Unintended Consequences of Advertising. J. Mark. 1986; 50(2): 18â36. JSTOR. Publisher Full Text\n\nPoushter J, Kent N: The Global Divide on Homosexuality Persists. Pew Research Center;2020.Reference Source\n\nRagusa AT: Social change and the corporate construction of gay markets in the New York Timesâ advertising business news. Media Cult. Soc. 2005; 27(5): 653â676. Publisher Full Text\n\nRead GL, Innis IJ, van Driel II , et al.: Mates or Married? Implications of Gender Composition and Physical Intimacy on Evaluation of Images Tested for Advertising. Commun. Res. Rep. 2019; 36(3): 220â230. Publisher Full Text\n\nRuggs EN, Stuart JA, Yang LW: The effect of traditionally marginalized groups in advertising on consumer response. Mark. Lett. 2018; 29(3): 319â335. Publisher Full Text\n\nSanz C: Playlist TV commercials in Spain. Anuncios de TV de España;2016.Reference Source\n\nSchmidt S, Stocker P: Omunicação, juventude e diversidade. Revista EletrÃŽnica Internacional de Economia PolÃtica Da Informação, Da Comunicação e Da Cultura. 2013; 15(3): 178â189.\n\nShinoda LM, Veludo-de-Oliveira T, Pereira I: Beyond gender stereotypes: The missing women in print advertising. Int. J. Advert. 2021; 40(4): 629â656. Publisher Full Text\n\nTaylor CR, Costello JP:The Social Identity Theory of Group Behavior. PsychoIogy of Intergroup Relations. Austin:1985; (Vol. 2, pp. 7â24).\n\nTaylor CR, Costello JP:Corporate Social Responsibility and the Portrayal of Minority Groups in Advertising. Handbook of Integrated CSR Communication. Springer;2017; (pp. 361â375). Publisher Full Text\n\nUm N-H: Does gay-themed advertising haunt your brand? Int. J. Advert. 2014; 33(4): 811â832. Publisher Full Text\n\nYu T-F: Class as a method to localise queer studies in Hong Kong. Gend. Place Cult. 2018; 25(2): 309â312. Publisher Full Text\n\nZmuda N: Ad campaigns are finally reflecting diversity of U.S. AdAge;2014.Reference Source"
}
|
[
{
"id": "167054",
"date": "22 Mar 2023",
"name": "Rafael Ravina-Ripoll",
"expertise": [
"Reviewer Expertise Happiness management",
"organizational comunication"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have enjoyed reading this paper. I appreciate that the author(s) focus on this issue: representations of gay men and lesbians collective is original and infrequent in literature. I appreciate the effort made to analyse how these collective are represented in ads. I believe research around this topic would be of interest for this Journal and could contribute to scholarly conversations around sexual diversity, media, pop culture and more.\nMy reading of the essay suggests that the author(s) are concerned only about the LGBT representations of ads but that they did only with a particular framework, Spain. The comparison between countries could be interesting in future, even if there are different approaches for the same advertisers but identifying adaptation in same countries, etc.\n\nOther suggestion or personal doubt, could the same topic be analysed in other kind of contents different to advertising, for example films, series or videoclips?\nI would be find a specific discussion in that section with the mirror theory. I am impressed with this theory linked with this topic so... it is a strength as theoretical contribution as a start point for further discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "167055",
"date": "23 Mar 2023",
"name": "Pedro A. Pereira Correia",
"expertise": [
"Reviewer Expertise Marketing and communication"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very original and interesting topical research study with a historic approach focused on Spain. I often read and contribute to studies related to gender image in advertising but it is not so usually the analysis of LGBT and other âspecial collectiveâ. Thus, thank you for the opportunity to review this essay. I wish the author(s) very well as they continue their important work.\nThe list of features used for the content analysis is very comprehensive and could be used to analyze ads in other contexts, specially providing international comparison. The paper is very well written and the findings are quite interesting.\n\nIn terms of improvement, I have the following suggestions:\nIn the abstract, the researchers mention that the goal of the ads has moved from a commercial angle to a more social function, however, this is not really emphasized in the discussion in contrast with the mentioned Mirror Theory. The theoretical implications could be interesting.\n\nIn a future line of research, could gender-fluid and other trends be included?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "167056",
"date": "05 Apr 2023",
"name": "Irene GarcÃa Medina",
"expertise": [
"Reviewer Expertise Marketing",
"Advertising"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article analyses how advertising and society interact with the gay and lesbian community and all the changes that happened in Spain from 1960 until 2021.\nThe methodology is adequate (a content analysis and a review of legislation and historical milestones).\nThe findings and conclusion are correct and the references used are good, both in number and in importance.\nFor all this, I consider this article of a very good quality and very important for the society and the academia.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-295
|
https://f1000research.com/articles/12-161/v1
|
10 Feb 23
|
{
"type": "Research Article",
"title": "Non-emergency medical transportation practice in Shanghai",
"authors": [
"Weidong Lu",
"Wei Xu",
"Long Shen",
"Wei Xu"
],
"abstract": "Background: To describe the current landscape of non-emergency medical transportation (NEMT) services in Chinaâs mainland, and analyze information obtained from a private NEMT company in Shanghai.\nMethods: With regards to the NEMT environment in Chinaâs mainland, we collected relevant NEMT policies from the websites of local Health Commissions, and collected business operating data from a Chinese enterprise information query tool, Qichacha. With regards to NEMT service in Shanghai, we analyzed operating data from 3426 trips by a professional NEMT company. Descriptive statistics were used to explain the characteristics of NEMT trips, and log-linear analyses were performed to compare the trips inside Shanghai with trips to/from other areas. Results: Of the 3426 trips in this study, there were 2962 trips inside Shanghai (86.5%) and 464 trips to/from other areas (13.5%), and the number of trips to/from each province was related to the distance to Shanghai. When comparing transportation types between trips inside Shanghai and to/from other areas, there was a significant difference (Ï2=144.87, p<0.001), with a significantly larger proportion of trips for discharge to/from other areas, and a significantly larger proportion of referrals in trips inside Shanghai. Over 50% of trips were to orthopedics-featured class A tertiary hospitals. Discussion and Conclusion: To lower the NEMT service costs and bring benefit to patients with a broader spectrum of diseases, medical insurance could completely or partially cover NEMT services, and companies could initiate rideshare services. Shanghai is an attractive place for patients, with the highest level of healthcare resources. Transportation combining high-speed trains and NEMT vehicles ensures patients who live further away can access health care in Shanghai.",
"keywords": [
"non-emergency medical transportation",
"transportation",
"healthcare access",
"Shanghai"
],
"content": "Introduction\n\nNon-emergency medical transportation (NEMT), a type of healthcare transportation, is an important social determinant. Before 2013, NEMT operated under local medical emergency centers in the Chinese mainland and occupied a huge amount of emergency transportation capacity. A 2010 statistical analysis in Shanghai showed that NEMT, including general transfers between hospitals and discharge transfers, surpassed 40% of the total emergency transportation business, leading to a relatively high ambulance rejection rate and slow departure rate.1\n\nIn 2013, the National Health Commission of the Peopleâs Republic of China issued a regulationâ âManagement Approach of Pre-hospital Medical Emergencyââclearly stating that pre-hospital emergency ambulances should not be used for non-emergency transportation. With this regulation, the burden on local medical emergency centers has fallen greatly; however, the demand for NEMT has surged fiercely due to the development of the economy, acceleration of population aging, implementation of a medical reform system with hierarchical diagnosis and treatment, and an increasing medicalânursing combination service.2\n\nCurrently, non-governmental sectors have been allowed to enter this field, and most provinces and cities have developed relevant policies and measures to help regulate and manage the business. By using a Chinese enterprise information query tool, Qichacha (https://www.qcc.com/), it was found that there were only 700 NEMT companies before 2020, with a surge in newly funded companies of 3000 in 2020 and 1000 in 2021, respectively. Although there was a huge increase in numbers, over 70% of companies have a registered capital of less than RMB 2 million and the current market cannot fully satisfy passenger needs.\n\nIn this paper, we aim to describe the current landscape of NEMT services and analyze information obtained from a private NEMT company in Shanghai. We have investigated the relevant policies of provinces and prefecture-level cities and operating environment, categories, and scales, and analyzed specific cases from a NEMT provider in Shanghai which utilizes the WeChat official account and mini-program to improve service delivery of healthcare transportation.\n\n\nMethods\n\nPolicy context\n\nFrom the websites of local Health Commissions on Chinaâs mainland, we collected relevant NEMT policies. By July 2021, 12 (38.70%) provinces and 35 (11.95%) prefecture-level cities had issued policies and regulations to support the market operation of NEMT. Most of these provinces and prefecture-level cities are more developed and located in the east part of China. With the prominent contradiction between supply and demand of NEMT, more province-level and city-level policies will be developed and improved.\n\nNEMT used to be a service provided by the local emergency medical centers (EMC) belonging to public institutions, and the 2013 policy âManagement Approach of Pre-hospital Medical Emergencyâ does not specify what kind of entities should conduct NEMT business and how it will operate. Most provinces and cities have explored their own business models, and 45 cities have been piloting NEMT services since 2013; more than half of the cities are from Guangdong, Jiangsu, and Shandong Provinces. In the way that 120 is used as an emergency call number throughout Chinaâs mainland, similarly, some cities or areas such as Shanghai, Tianjin, Nanjing, Qingdao, Hangzhou, etc., used unified NEMT service numbers.\n\nOperating environment\n\nThere were over 5000 companies running NEMT businesses up to July 2022, according to Qichacha. Most NEMT companies were founded in 2020 and 2021, with about 60% being founded in 2020. Over 70% of these companies belong to the national industries of health and social work, and the remaining companies are widely distributed in the industries of resident services, repairing and other services, transportation, warehousing and postal services, and wholesale and retail services.\n\nGuangdong province owns half of the NEMT companies, followed by Shandong province (around 10%) and Jiangsu province (around 8%).\n\nAfter analysis, we identified three core-operating types of NEMT services: 1) a NEMT department under the local EMC; 2) a NEMT company running independently; and 3) a unified NEMT service platform integrating local private companies.\n\nType 1: a NEMT department under the local EMC. In some cities, such as Shanghai and Tianjin, the NEMT department was created as a branch of the local EMC. The critical feature of this type is that NEMT is owned and operated by nonprofit local EMCs, and NEMTâs special calling line is set up under 120 command and dispatch centers.\n\nType 2: a NEMT company running independently. NEMT companies can be state-owned enterprises, private medical institutions, or other private enterprises. These companies can operate independently with their own service calls, APPs, mini-programs, etc. Some provinces or cities, such as Ningxia Hui Autonomous Region, have allowed the operation of private enterprises approved by local administration for market regulations; however, others, such as Hubei Province, need them approved by not only local administration for market regulations but also health commissions as well. Some provinces or cities have also ordered that NEMT personnel should accept the training provided by the local medical emergency center and obtain a first-aid certificate.\n\nType 3: a unified NEMT service platform integrating local private companies. The unified NEMT service platform, such as the NEMT Service Platform of Qingdao, has incorporated qualified NEMT companies, uses consistent service specifications and charging systems, and provides one unified service phone number, APP, official account, and mini-program.\n\nNEMT service environment in Shanghai\n\nShanghai is the most advanced city in China, and is a city with the highest degree of population aging. According to statistics, the population of residents aged 65 and above has exceeded 4 million, accounting for 16.3% of the total population (https://www.shanghai.gov.cn/nw12344/20210518/001a0cef127c499eb381fa8dc3208e95.html).\n\nIn 2020, the total number of medical visits to Shanghai medical institutions was over 240 million, with outpatient and emergency visits reaching more than 216 million, and inpatient surgeries exceeding 3 million (http://wsjkw.sh.gov.cn/tjsj2/20210426/eb18f046dea54e129bd3acb3dfbc95cc.html). As the largest destination for medical treatment in China, Shanghai has accepted approximately 0.14% of medical visits by patients from other regions. According to the statistics from the National Health Commission, there were more than 370 thousand patients outside Shanghai in 2016.3\n\nShanghai has over 980 ambulances and 175 first-aid stations by 2020 (http://wsjkw.sh.gov.cn/rdhy/20200428/6a533e9e7cd24f56a86cc0e1ac8ee470.html). The average service radius has been shortened to 3.5 kilometers, and the average first-aid response time was within 12 minutes in April 2021. In 2020, Shanghai EMC completed over 19 million kilometers of pre-hospital first aid throughout the year, with 921 thousand first-aid deliveries.\n\nIn 2018, Shanghai EMC set up a NEMT service line for the public, with 30 NEMT vehicles from the beginning and 50 by the end of 2021, providing around 100 thousand NEMT trips per year (accounting for about 12â16% of emergency services). However, compared with around 300 thousand incoming calls a year for NEMT needs, there is still a considerable gap between supply and demand.\n\nFurthermore, NEMT provided by Shanghai EMC serves only residents who live in the Shanghai area. A certain number of patients living outside Shanghai have to depend on other NEMT vehicles.\n\nWe conducted this study at a professional NEMT company, which is the first government-approval private NEMT company in Shanghai. The company received the NEMT service approval from a district health commission of Shanghai in December 2019 and began to operate in August 2020. After nearly half a year of trial operation with three NEMT vehicles, the company became officially operational in March 2021 with 10 NEMT vehicles, and all the crew members were trained and certified by Shanghai EMC.\n\nThe operation database of the company consists of about 5000 trips with longitudinal data, including transportation time, departure address, destination address, hospital type, and transportation type.\n\nThe data for this study were collected from March 2021 until February 2022, with a total of 3426 trips. The data were populated into Excel first, and then transferred to and checked in SPSS version 21 (RRID:SCR_002865). Descriptive statistics were used to explain the characteristics of NEMT trips. Log-linear analyses were performed to check the difference in the transportation type between groups of trips inside Shanghai and trips to/from other areas.\n\n\nResults\n\nOf the 3426 trips in this study, there were 2962 trips inside Shanghai (86.5%) and 464 trips to/from other areas (13.5%). Departure or destination locations of trips to/from other areas covered nine provinces, with 315 trips (9.2%) to/from Jiangsu province, 77 trips (2.2%) to/from Zhejiang province, and 42 trips (1.2%) to/from Anhui province (Figure 1). The number of trips to/from each province is related to the distance to Shanghai. There were also 53 trips to/from Shanghai railway station or airport. That means some patients from other areas used not only NEMT services but also long-distance public transportation such as flights or trains to travel to/from medical institutions in Shanghai.\n\nWe grouped transportation types into discharge, admission, referral, and others. The leading transportation type of trip was referral (1881, 54.9%), followed by discharge (1312, 38.3%). The smallest portion was others (59, 1.7%), which included trips where neither the departure nor destination location was medical institutions, such as trips from homes to nursing institutions, stations to homes, etc. (Table 1). With regards to trips inside Shanghai, referral accounted for most trips (1736, 47.6%), about 1018 trips were for discharge (34.4%), 157 (5.3%) were for admission, and about 51 (1.7%) trips served other purposes; In trips to/from other areas, most trips were for discharge (294, 63.4%), followed by 145 (25.2%) for referral, 17 (3.7%) for admission, and 8 (1.7%) for others.\n\nWhen comparing transportation types between trips inside Shanghai and to/from other areas, there was a significant difference (Ï2=144.87, p<0.001). According to log-linear analysis, transportation types of discharges and referrals were the most significantly different types, with a significantly larger proportion of trips for discharge to/from other areas, and a significantly larger proportion of referrals for trips inside Shanghai.\n\nThe transportation type of referral has been further classified, as referral has different modes. For trips inside Shanghai, we classified referrals into downward referral, upward referral, and same-level referral. Downward referral often means patients in an upper-level hospital who have reached the convalescent or stable stage and can be transferred to a lower-level hospital. Upward referral refers to patients with critical or complicated diseases in a lower-level hospital who can be transferred to an upper-level hospital. Same-level referral refers to patients who may be transferred between same-level hospitals to different departments or hospitals with different specialties. There were 1736 referral trips, and most of the trips were downward referral (1410, 81.2%), while trips in upward referral and same-level referral shared almost the same numbers with 166 (9.6%) and 160 (9.2%), respectively.\n\nIn the group of trips to/from other areas, we classified referrals into referrals to Shanghai and referrals from Shanghai, for it is difficult to evaluate the medical level of hospitals between different areas. Most of the trips in referral type were referrals from Shanghai (136, 93.8%), and there were only eight trips in referrals to Shanghai (5.5%) (Table 2).\n\nClass A tertiary hospitals (3A hospitals) are the highest level medical institutions classified in accordance with the Administrative Measures for Hospital Classification in China. Admissions, discharges and referrals are transportation types whose departure or/and destination locations are hospitals. A total of 3367 trips were the types of admission, discharge and referral, and 2558 (76.0%) trips involved 3A hospitals (Table 3).\n\nIn trips inside Shanghai, there were 2189 (75.2%) trips to/from 3A hospitals, of which 82 (52.2%) were trips for admission from homes to 3A hospitals, 686 (67.4%) were trips for discharge from 3A hospitals to homes, and 1421 (81.9%) were trips for referrals to/from 3A hospitals. In trips to/from other areas, there were 369 (80.9%) trips to/from 3A hospitals, of which 16 (94.1%) were trips for admission from homes to 3A hospitals, 231 (78.6%) were trips for discharge from 3A hospitals to homes, and 122 (84.1%) were trips for referrals to/from 3A hospitals.\n\nIn trips to/from other areas, there were 24 (96.0%) trips to 3A hospitals in Shanghai including 17 trips for admission and 8 trips for referral to Shanghai. One trip was to a hospital outside Shanghai (Table 3).\n\nSix 3A hospitals with the highest number of trips are listed in Figure 2. There were 2366 trips to/from the six hospitals, accounting for 70% of total trips for admission, discharge and referral. The first three 3A hospitals with the highest number of trips in both groups of trips inside Shanghai and trips to/from Shanghai were the same: Shanghai 6th Peopleâs Hospital, Zhongshan Hospital, and Changzheng Hospital, and the proportion of trips to/from them surpassed half of the total trips involving hospitals (64.7%).\n\nTrips to/from the Shanghai 6th Peopleâs Hospital were the largest proportion (42.8%). The most featured department in both Shanghai 6th Peopleâs Hospital and Changzheng Hospital was orthopedics. When interviewing NEMT dispatchers and escorts, we received the experienced information that about 80â90% of the patients were for orthopedics, followed by cardio-cerebrovascular disease and neurology.\n\n\nDiscussion\n\nMany countries have mature NEMT markets with national or local service standards and regulations. NEMT services are quite different among these countries. In the USA, NEMT often means rides for Medicaid-eligible individuals to and from the doctorâs office, the hospital, or another medical office for Medicaid-approved care (https://www.cms.gov/Medicare-Medicaid-Coordination/Fraud-Prevention/Medicaid-Integrity-Program/Education/Non-Emergency-Medical-Transport), and NEMT vehicles might be wheelchair vans, taxis, stretcher cars, and buses, depending on the patientâs needs and State rules (https://www.cms.gov/Medicare-Medicaid-Coordination/Fraud-Prevention/Medicaid-Integrity-Education/Downloads/nemt-booklet.pdf). In Australia, NEMT generally refers to trips provided for persons to or from medical services using a stretcher-carrying vehicle, with specialist clinical care or monitoring, and the NEMT rides should be authorized by the State Ambulance Service (https://www.qld.gov.au/seniors/transport/transport-assistance/non-emergency-medical-transport, https://www.health.tas.gov.au/professionals/non-emergency-patient-transport#eligibility-requirements-for-nept-services, https://www.legislation.vic.gov.au/in-force/acts/non-emergency-patient-transport-act-2003/014). In China, the definition of NEMT varies slightly among provinces and cities, however NEMT vehicles are similar among provinces and cities. NEMT was defined as trips from medical institutions to non-medical institutions requiring special vehicles without any medical actions in the report âImplementation Opinions on Improving the Classification of the Pre-hospital First Aid System in Shanghaiâ (http://wsjkw.sh.gov.cn/yzgl3/20180815/0012-59762.html). The special vehicle often means disabled transfer vehicles such as wheelchair or stretcher-carrying vans. NEMT vehicles used in China are consistent with those in Australia.\n\nNEMT has become a rapidly growing industry since separating from government-owned emergency institutions. Although quite a few provinces or cities issued NEMT-related policies, there were no NEMT standards to regulate the business before 2022. The quality of NEMT services varied among providers, with various internal standards if they existed.\n\nPrevious research has shown that the percentage of NEMT in total trips of local EMC varied from over 10% to 60%, and it was about 30% in Shanghai.5 Medical institutions in Shanghai completed 272 million visits in 2021 including over 2.4 million visits from other provinces (http://tjj.sh.gov.cn/tjgb/20220314/e0dcefec098c47a8b345c996081b5c94.html). Therefore, there is a big market for NEMT services. Although Shanghai EMC has provided 50 ambulances for NEMT services, with 100 thousand trips per year, compared with 921 thousand first-aid transportations, there is a substantial gap to meet patientsâ NEMT needs. Private institutions have a great opportunity to enter this sector.\n\nWe have analyzed the operation data of the biggest private NEMT company in Shanghai, and the results would be valuable for promoting NEMT service capabilities in Shanghai.\n\nThe company completed a total of 3426 trips from March 2021 to February 2022, and the figure accounted for about only 3.4% of those by Shanghai EMC. The operation is not at full capacity for the following reasons: 1. residents are prone to trust the government-owned NEMT department; 2. the NEMT department in Shanghai EMC with government subsidies provides a much more competitive price; and 3. NEMT is still a new service that lacks public awareness.\n\nShanghai has always been the city with the highest influx of patients from other provinces, and tertiary hospitals accepted over 2.4 million patients outside Shanghai in 2019, accounting for 40.12% of total migrant patients in China. Among 3426 total trips, there were 2962 trips inside Shanghai (86.5%) and 464 trips to/from other areas (13.5%). The top three provinces with the largest number of trips to/from outside Shanghai were Jiangsu, Zhejiang, and Anhui, and the finding is consistent with statistical results from National Health Commission.3 Other provinces included Jiangxi, Fujian, Henan, Shandong, Hebei, and Hainan. The number of trips between other provinces and Shanghai is related to the distance, and Jiangsu has the most cities closest to Shanghai, followed by Zhejiang and Anhui. The longest trip was to Hainan, a distance of more than 1300 miles.\n\nThe leading transportation type of trip was referral (1881, 54.9%), followed by discharge (1312, 38.3%), and admission (174, 5.1%). The smallest proportion was others (59, 1.7%). Over 90% of the trips were for referral and discharge. The number of trips for referral exceeded those for discharge, and the ratio of referral to discharge (54.9:38.3, 1.43) was consistent with previous figures from transportation statistics of Shanghai EMC in 2010 (23.4:17.9, 1.31).4 This ratio was quite different from those in other cities such as Qingdao (14.0:29.6, 0.48), Suzhou (34.8:65.2, 0.53), and Wuhan (21.4:54.3, 0.39) between 2014 and 2018.2,5,6 In the latter cities, the number of trips for referral was far below that for discharge.\n\nAlthough China has advocated tiered diagnosis and treatment since 2006, when âGuidelines on the Development of Urban Community Health Servicesâ was issued by the State Council (http://www.nhc.gov.cn/wjw/gfxwj/201304/df3e35e26b3a4f5987bd898ddce70404.shtml), patients are more willing to visit tertiary hospitals and be discharged from them when completely recovered, as tertiary hospitals provide a higher quality of medical resources and better medical services, leading to more NEMT trips for discharge than for referral. The ratio results in Qingdao, Suzhou and Wuhan have revealed patientsâ choices during 2014â2018. With the further implementation of âGuidelines on Promoting the Construction of Hierarchical Medical Systemâ issued by the State Council in 2015 (http://www.nhc.gov.cn/cms-search/xxgk/getManuscriptXxgk.htm?id=c30041e1016a427f9477774c9e864eb4), trips for referral might gradually increase.\n\nUnlike many other cities in China, Shanghai has begun to create medical alliances since 2003, and most of the 3A hospitals established hospital groups or consortiums with medical institutions in 10 districts and suburban counties around 2006. Referrals among medical alliances have been much more frequent than in other cities. Efficient implementation of medical alliances reduces the length of stay and promotes bed turnover rates. According to statistics in 2018, the average length of stay in tertiary hospitals in Shanghai was 6.4 days, far below the figure in tertiary hospitals nationwide (9.1 days). This may explain the reason why there are more NEMT trips for referral than for discharge in Shanghai.\n\nThere was a significant difference between groups of trips inside Shanghai and trips to/from other areas, with more trips for referral in Shanghai, and more trips for discharge to/from other provinces. Part of the reason was that most patients from other provinces received therapy from 3A hospitals in Shanghai then transferred to a rehabilitation hospital within 3A hospital alliances to achieve full recovery before going back home, and these data were grouped into trips for referral inside Shanghai.\n\nThere were 2558 trips with 3A hospitals as departure or destination locations, accounting for 76.0% among 3367 trips for admission, discharge, and referral. With regards to trips for admission, about half of the Shanghai patients visited 3A hospitals, while almost all the patients from other provinces were admitted to 3A hospitals in Shanghai. Although 3A hospitals are of the highest level among all the medical institutions in China, medical quality is quite different among 3A hospitals across the nation. Top level 3A hospitals are mainly located in Beijing, Shanghai, Guangzhou and some coastal regions, and 11 of the best 50 3A hospitals are in Shanghai. Therefore, patients from other provinces would rather spend more on transportation and accommodation to seek the best medical treatment in Shanghai.\n\nAlthough the company did not collect trip purposes at this time, we can infer from the hospital specialty and the experience of dispatchers and escorts that the three leading primary purposes for patients who requested NEMT were for the departments of orthopedics, cardio-cerebrovascular, and neurology disease. This finding is quite different from that of a study in the USA.7 The research in the USA found that the leading primary trip purposes were for dialysis, doctor visits, substance use, and mental health. Over 77% of round trips were for travelers who required no assistance devices, and no more than 4% required a stretcher. Almost all the NEMT vehicles in China are stretcher vans, and patients themselves pay for transportation expenses; as a result, only disabled passengers or those who cannot move request NEMT services, while the types of NEMT vehicles in the USA are much broader and passengers have their transportation fee paid by Medicaid. Therefore, any patients with Medicaid can use NEMT to/from medical institutions.\n\nWe must acknowledge the limitations associated with this study that hindered our ability to achieve more valuable and accurate results. First, it is important to note that we extracted data from a newly operated company and their data repository was not complete enough to reveal the complete transportation characteristics. The analyses are somewhat limited as the patientsâ age, gender, trip purpose, required equipment, and trip distance were not available. Previous research has shown that one of the priorities for an NEMT vendor is data driven. Inadequate data collection may lead to unknown patient profiles and needs, and unknown NEMT influence on patient satisfaction, hospital bed turnover, clinical results, and financial outcomes.8\n\nWithout patient profiles at this time, we can only compare trips inside Shanghai with trips to/from Shanghai, as patients from other provinces might transfer between hospitals in Shanghai for further rehabilitation. Therefore, the data could not accurately represent the patients from other provinces.\n\nGiven the findings and limitations, there are some practical implications for both policymakers and NEMT companies. First, standardization of the NEMT process would be both valuable and easy to implement as an intervention for reducing risk and increasing efficiency.9 NEMT practitioners have begun to realize the importance of developing NEMT-related standards, and two social organization standards for NEMT service specifications have been issued since May 2022 (http://www.ttbz.org.cn/StandardManage/Detail/65630/; http://www.ttbz.org.cn/StandardManage/Detail/60985/). It may be a good beginning to initiate a series of standards for process, vehicles, personnel, etc.\n\nSecond, NEMT in China is a service entirely paid for by the patients, and, because the expense is much higher than a taxi service, only patients with severe fractures, neurological diseases, or cardiovascular diseases who are limited in mobility use NEMT; however, there are other health-care needs that are prevalent among people with transportation disadvantages including dialysis, prenatal care, cancer treatment, and mental health and substance-use treatment.10 Medical insurance that partially covers NEMT would benefit patients with a broader spectrum of diseases by allowing them to access health care more efficiently and smoothly.\n\nThird, to lower the NEMT service costs, companies could initiate rideshare services. In the USA, although NEMT is an important Medicaid benefit, it confronts service and funding cuts, strict enrollment, and various restrictions. Rideshare-based medical transportation (RMT) has become an NEMT alternative that may provide cost reduction and flexibility for riders. Rideshare companies such as Uber and Lyft extended their business to include NEMT around 2016, and more states accept rideshare services as NEMT alternatives.11,12 In China, rideshare services have not entered NEMT yet, and operating RMT may be hindered by two main reasons. The first reason is that most NEMT vehicles are stretcher vans to take patients with mobility disabilities, and they may have no extra space to pick up more passengers. Even Rideshare services in the USA do not provide rides to passengers using wheelchairs/stretchers. The second reason is that, as most passengers are also patients, they might feel upset sharing a vehicle with other patients. To be initiated, RMT services need to extend vehicle types or revise current vehicles available for more passengers, and arrange shared passengers according to their disease types, avoiding passengers having to share vehicles with patients with certain diseases.\n\nFourth, almost all the patients from other provinces go to visit 3A hospitals in Shanghai for it provides the highest-level medical diagnoses and treatments; however, the best medical resource has been accessed mostly by patients from surrounding areas, and patients from many inland areas can hardly gain access because of the cost burden and transportation barriers. With all the difficulties, patients with serious or complicated diseases would like to find the best medical hospitals. The rapid development of high-speed trains with much lower expenses than flights makes it possible for patients who live far away from Shanghai to be admitted to medical hospitals in Shanghai. High-speed trains significantly reduce transportation time, and NEMT companies could extend transportation to combine high-speed trains with NEMT vehicles; that is, at departure locations, NEMT vehicles could bring patients to the high-speed train station, and escorts could help patients with wheelchairs/stretchers into carriages and through the train transportation. After arriving at the destination station, escorts could help deliver patients to NEMT vehicles and finally bring patients to destination locations. The NEMT company in this study has begun to operate high-speed trains plus NEMT services and successfully finished one Shanghai to Beijing transportation business within 5.5 hours, compared with more than 13 hours with NEMT vehicles only.\n\n\nConclusions\n\nNEMT is a rapid growing industry serving a critical role for patients with mobility disabilities to/from medical institutions. This study reveals the contextual environment associated with the operation of NEMT, providing a more complete picture in understanding utilization of NEMT, and analyzes differences for NEMT utilization between trips inside Shanghai and to/from other provinces.\n\nIt also provides suggestions to reduce transportation barriers by lowering NEMT costs and extending delivery mode to high-speed trains plus NEMT services. More research is needed to follow the results of RMT practice and high-speed trains plus NEMT services, and extend NEMT to more vulnerable populations.\n\nThe authors did not preregister the research at an independent registry.",
"appendix": "Data availability\n\nFigshare: Non-emergency medical transportation practice in Shanghai. https://doi.org/10.6084/m9.figshare.21555168.v1. 13\n\nThe project contains the following underlying data:\n\n- Trips to or from 3A hospitals.xlsx\n\n- Trips to or from other provinces.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nZhao HQ, Xie J: Discuss on the Category ambulance service of pre-hospital emergency system in Shanghai. China Journal of Emergency Resuscitation and Disaster Medicine. 2011; 6(5): 396â398.\n\nRan P, Lin AJ, Bie XY, et al.: Analysis and discussion on non-emergency transportation service mode and current situation. China Journal of Emergency Resuscitation and Disaster Medicine. 2020; 15(7): 795â799.\n\nNational Health Commission of the Peopleâs Republic of China: National Report on the Services, Quality and Safety in Medical Care System 2020. Beijing:Scientific and Technical Documentation Press;2021-10-01.\n\nZhao HQ, Xie J: Discuss on the Category ambulance service of pre-hospital emergency system in Shanghai. China Journal of Emergency Resuscitation and Disaster Medicine. 2011; 6(5): 396â401.\n\nZhou ZX, Yang QY, Wang DY, et al.: Exploration of non-emergency medical transfer service in Suzhou. Jiangsu Health System Management. 2018; 29(5): 605â607.\n\nYan M, Ao SS, Jiang WX, et al.: Investigation of demand of non-emergency in pre-hospital emergency in Wuhan. Chin. J. Mod. Nurs. 2016; 22(26): 3820â3821.\n\nSmith ML, Prohaska TR, MacLeod KE, et al.: Non-Emergency Medical Transportation Needs of Middle-Aged and Older Adults: A Rural-Urban Comparison in Delaware, USA. Int. J. Environ. Res. Public Health. 2017 Feb 10; 14(2): 174. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLyons PG, Ramsey BA, Welker M, et al.: Implementation of a non-emergent medical transportation programme at an integrated health system. BMJ Health Care Inform. 2021 Sep; 28(1): e100417. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHains IM, Marks A, Georgiou A, et al.: Non-emergency patient transport: what are the quality and safety issues? A systematic review. Int. J. Qual. Health Care. 2011 Feb; 23(1): 68â75. Epub 2010 Dec 1. PubMed Abstract | Publisher Full Text\n\nChen KL, Brozen M, Rollman JE, et al.: How is the COVID-19 pandemic shaping transportation access to health care? Transp Res Interdiscip Perspect. 2021 Jun; 10: 100338. Epub 2021 Mar 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEisenberg Y, Owen R, Crabb C, et al.: Rideshare transportation to health care: evidence from a Medicaid implementation. Am. J. Manag. Care. 2020 Sep 1; 26(9): e276âe281. PubMed Abstract | Publisher Full Text\n\nChaiyachati KH, Hubbard RA, Yeager A, et al.: Rideshare-based medical transportation for Medicaid patients and primary care show rates: a difference-in-difference analysis of a pilot program. J. Gen. Intern. Med. 2018 Jun; 33(6): 863â868. Epub 2018 Jan 29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFigshare: Non-emergency medical transportation practice in Shanghai. DOI: 10.6084/m9.figshare.21555168.v1"
}
|
[
{
"id": "163105",
"date": "24 Feb 2023",
"name": "Souad SASSI BOUDEMAGH",
"expertise": [
"Reviewer Expertise Project management-architecture and urban planning-Higher Education"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors are tackling an important social topic related to healthcare transportation. As highlighted, NEMT is a transportation service that helps individuals who require transportation to medical appointments, but do not have access to or cannot use conventional transportation options. In the introduction, authors provided the context of the study, and stated its aim.\nThe authors have appropriately explained the meaning, relevance and importance of the results and also  considered the limitations to the study. The discussion section provides a comprehensive overview of NEMT services in various countries and highlights the differences in the definitions, regulations, and vehicle types used in NEMT. The authors also discuss the growing NEMT industry in China, which has recently separated from government-owned emergency institutions, and the opportunities for private institutions to enter this sector.\nThe authors then present the operation data of the biggest private NEMT Company in Shanghai and provides valuable insights into the current state of NEMT services in the city. The low percentage of trips completed by the private company compared to the government-owned NEMT department is attributed to residents' trust in the latter, the competitive pricing, and the lack of public awareness of NEMT services.\nThe author also analyzes the trips made by the private NEMT Company and highlights the leading transportation types of trips, the provinces with the largest number of trips, and the distance covered.\nHowever, policy context and NEMT service environment in Shanghai description are overloading the Method section. In addition, even if the authors made an accurate statement of the studyâs major results in the discussion section, authors are moving from their study results to the context description, historical data and facts, and vice versa. The retrospective should be in the context description, since the study has been conducted from March 2021 until February 2022.\nThe Conclusion is too short however; it briefly summarizes the key findings of the study and suggests areas for further research and potential solutions to reduce transportation barriers for patients with mobility disabilities.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9416",
"date": "02 Mar 2023",
"name": "Weidong Lu",
"role": "Author Response",
"response": "Thank you very much for your review in so much detail. We read it carefully and completely agree with your comments. We would revise the paper according to your suggestions if we have chance. Thank you again."
},
{
"c_id": "9454",
"date": "17 Mar 2023",
"name": "Weidong Lu",
"role": "Author Response",
"response": "Thank you very much for your nice comments and suggestions on the manuscript: Non-emergency medical transportation practice in Shanghai. We revised our paper carefully and hope the concerns have been addressed adequately in this revision. However, policy context and NEMT service environment in Shanghai description are overloading the Method section. In addition, even if the authors made an accurate statement of the studyâs major results in the discussion section, authors are moving from their study results to the context description, historical data and facts, and vice versa. The retrospective should be in the context description, since the study has been conducted from March 2021 until February 2022. Response: Thank you very much and we agree with you. In the revision, we put policy context and NEMT service environment in Shanghai description in a new part: Background under Introduction. In the discussion section, we moved some context description, historical data and facts into Background part, and leave some description which we think will explain the results in Discussion part. The Conclusion is too short however; it briefly summarizes the key findings of the study and suggests areas for further research and potential solutions to reduce transportation barriers for patients with mobility disabilities. Response: Thank you very much for your suggestion. We made some revisions on Conclusion part."
}
]
}
] | 1
|
https://f1000research.com/articles/12-161
|
https://f1000research.com/articles/11-1074/v1
|
21 Sep 22
|
{
"type": "Study Protocol",
"title": "Physiotherapy interventions for head and trunk control in children with developmental disabilities: A scoping review protocol",
"authors": [
"Shristi Shakya",
"Shradha S. Parsekar",
"Selvam Ramachandran",
"Shamantha Madapura S.",
"Harikishan Balakrishna Shetty",
"Dana Anaby",
"Sivakumar Gopalakrishna",
"V. S. Venkatesan",
"Bhamini Krishna Rao",
"Shristi Shakya",
"Shradha S. Parsekar",
"Selvam Ramachandran",
"Shamantha Madapura S.",
"Harikishan Balakrishna Shetty",
"Dana Anaby",
"Sivakumar Gopalakrishna",
"V. S. Venkatesan"
],
"abstract": "Background: Head and trunk control is prerequisite skill that maximizes engagement and participation in oneâs environment by integrating vision, oromotor skill, arm control and respiration. Various physiotherapy and technology-based interventions have been utilized to facilitate head and trunk control in children with developmental disabilities. This scoping review is planned to map and summarize existing studies from the scientific literature on physiotherapy and technology-based interventions for head and trunk control in children with developmental disabilities. Methods: The scoping review will utilize the Joanna Briggs Institute scoping review methodology. The review will cover studies including children and adolescents aged between two years and 17 years 11 months 29 days, with developmental disabilities where in child finds difficulty aligning head and trunk in sitting position, against gravity, for more than a minute. We will include randomized controlled trial (RCT), non-RCT, quasi-experimental trial, and systematic reviews that have employed physiotherapy and technology-based interventions. Database-specific search strategy will be used to search records in Medline (PubMed and Web of Science), Embase, Scopus, CINAHL, PEDro, and Cochrane Library. Additionally, various grey literatures and clinical-trial registries will be searched. Two reviewers, independently, will screen and extract the data. Tables and visual representations will be utilized to present the extracted data. Registration details: The protocol has been registered in Open Science Framework, DOI:Â 10.17605/OSF.IO/B3RSU (22nd August 2022)",
"keywords": [
"Head control",
"physiotherapy",
"postural control",
"technology-based intervention",
"trunk control"
],
"content": "Introduction\n\nDevelopmental disability can be defined as, âas a severe and chronic disability that manifests before age 22, is attributable to a mental and/or physical impairment, results in substantial functional limitations in three or more major life activities and reflects a need for a combination and sequence of special, individualized services that are of extended duration or lifelongâ.1 In 2016, 52.9 million children under the age of five had developmental disability.2 Developmental disability encompasses wide range of conditions such as, Global Developmental Delay (GDD) accounting for 5-15% of prevalence worldwide,3 Cerebral Palsy (CP) affecting 1.5 to 4 out of every 1,000 live births across the world,4 Down Syndrome (DS) accounting 10 to 11 out of every 10,000 live births worldwide,5 and Spinal Muscular Atrophy (SMA) with a global incidence of 1 in 12,000 live birth.6\n\nThere are many symptoms of children with developmental disabilities, among them the one that requires immediate attention is child facing difficulty to align head and trunk in a sitting position, against gravity. Head control is the primordial motor skill essential for the development and acquisition of higher-level motor abilities besides their contributory role of integrating vision, regulating breathing patterns, and deglutition. These activities are essential for a child's independence in everyday life, and eventually, quality of life (QOL).7,8 Trunk control, a component of the postural control framework, involves trunk stability and mobility. During the first year of life, the development of trunk control prepares the child to establish anti-gravity motor control to facilitate independent environmental participation, which is commonly impaired in children with developmental disabilities. A stable trunk aids in orienting the child to his/her environment, allowing the child's social, cognitive, and communicative abilities to develop.9\n\nGiven the fact that children with developmental disabilities require regular treatment sessions, therapists may find it challenging to engage the children throughout the rehabilitation program. Therefore, there is a need to develop unique ways and tools to encourage children perform exercises that are both affordable and acceptable. Children with disabilities often gets tired or bored performing monotonous exercises hence, (advanced) technology-based interventions may help build an interactive and child-engaging rehabilitation approaches7,10,11 and/or may complement existing strategies. Hence, there is a need to incorporate enjoyable and interactive mode of rehabilitation for the children with developmental disabilities, to involve the children in the rehabilitation session.\n\nWith this concept, CanChild developed the âF-words in Childhood Disabilityâ concept, consisting of âFunction,â âFamily,â âFitness,â âFun,â âFriends,â and âFuture,â and is a conceptualization and modification of the World Health Organizationâs (WHO) (2001) the International Classification of Functioning, Disability, and Health (ICF) framework,12 with the goal of incorporating âfun elementâ in exercises to make the sessions enjoyable and interactive for the child.12,13 There are many rehabilitation interventions utilized for improving head and trunk control in children with developmental disabilities including the application of multiple physiotherapy (active and passive) and technology-based interventions (manual and digital), which can augment these outcomes by facilitating enhanced interactions and engagement during therapy sessions. The data file 1 in the Extended data highlight some of the physiotherapy and technology-based interventions.13,14\n\nIn literature, most studies focus on standing and balancing abilities of children with developmental disabilities. However, it is impossible to attain stable balance and posture without addressing the core component involved in such abilities, namely, head and trunk control. Studies suggest that newer devices can be utilized to improve childâs motor control, and children find this kind of practice motivating and fun.7,10,11 Studies have also utilized many treatment approaches listed under data file 1.14,15,17,18 Most of the study interventions have predominantly focused on impairments and activity limitations in reporting the facilitation of head and trunk control.\n\nTherefore, we intend to utilize physiotherapy and technology-based intervention for developing head and trunk control in children with developmental disabilities, which carries element of fun, variability, interactivity, engagement, motivation, and sense of accomplishment to the children during their therapy session. Moreover, head and trunk control can be considered as a foundation to integrate vision, oromotor skill, arm control, and respiration, ultimately aiding in improving QOL which is broadly described in the âUnited Nations Sustainable Developmental Goals 3 â Good Health and Well-beingâ,19 as well as, the use of available interventions to reduce the burden of persons with disability may help in improving the economic and social burden of the country as mentioned in the âUnited Nations Sustainable Developmental Goals 10 â Reduced Inequalitiesâ.20\n\nA preliminary literature search on the topic showed that most interventions were limited to medical (medicines, surgery, bone-and-tendon lengthening procedures) or re/habilitation management, focusing grossly on improvement of function and strength in extremities, without paying much attention to underlying measures of postural control.21The components of postural control have been identified as construct in relation to the outcome measures, such as, static, and dynamic stability, functional stability limit, cognitive influences, verticality, and reactive postural control. Only few technology-based interventions such as Virtual Reality (VR),21 Interactive Computer Play (ICP),10 Robotics22 have been used in re/habilitation practice. However, most of the interventions were targeted to gross motor functions rather than focusing on the construct.23\n\nFurthermore, a review aimed at identifying and evaluating physical therapy studies in CP children found that majority of studies intended to reduce âimpairments and activity limitationsâ, with little attention directed to âparticipation and environmental factorsâ.16 Another review aimed to understand and describe literature addressing effect of altered postural and segmental trunk control on sitting among children with CP. However, it did not focus on strategies to improve postural and segmental trunk control, and interventional studies were excluded.24 No scoping review exists that summarizes studies assessing physiotherapy and technology-based interventions for postural control measuring core element of gross motor skill acquisition. Most of the reviews were restricted to geographical locations and selective interventions, and none of the reviews focused on the core component of postural and gross motor control i.e., head and trunk control. Therefore, this scoping review is planned to address these gaps by answering following questions.\n\n\n\n1. What physiotherapy and technology-based interventions are available that assessed head and trunk control and gross motor functions in children with developmental disabilities?\n\n2. What studies are available that measured effectiveness of the physiotherapy and technology-based interventions on head and trunk control and gross motor functions in children with developmental disabilities?\n\nThe first question will map available physiotherapy, which also includes technology-based interventions that assessed head and trunk control in children with developmental disabilities. Through second question, we want to summarize available evidence and gaps in the studies assessing intervention and selected outcomes, which may help research community to either design robust interventional studies (if gap exists) or plan a systematic review (based on availability of evidence). This may also help funders in prioritizing research topics while calling for research proposals.\n\n\nProtocol\n\nThe proposed scoping review will be conducted in accordance with the Joanna Briggs Institute methodology for scoping reviews.25 The protocol has been registered in Open Science Framework, DOI: 10.17605/OSF.IO/B3RSU.26 In the case that the proposed protocol is amended, we will detail the change and provide justifications in the final review.\n\nEligibility criteria\n\nParticipants: Studies conducted among children between two years to 17 years 11 months and 29 days, who have developmental or neuromotor impairment, with difficulty to align head and trunk in sitting position, against gravity will be included. This difficulty in head and trunk control is frequently seen in conditions such as developmental delay, CP, DS and SMA. Developmental disabilities may include functional limitations of more than three areas of life activities, such as respiration, deglutition, bimanual activities, and bipedal walking. Studies conducted among children and adolescents who have co-morbidities such as Cortical Visual Impairment (CVI), hearing disability, intellectual disability, terminal illness receiving hospice care, and having a history of seizure disorders will be excluded as some of the physiotherapy interventions may be contraindicated among these children and these children may need medical management. Furthermore, this review will exclude population having acquired conditions affecting head and trunk control.\n\nConcept: We will include studies that assessed effectiveness of physiotherapy including technology-based interventions, which are listed, but not restricted to, the ones mentioned in data file 1 in the Extended data. However, the focus of the interventions should be on head and trunk control. The physiotherapy interventions could be varied and classified as âactive interventionsâ, which includes usage of muscles or joints voluntarily by the participants on command, and âpassive interventionsâ, which includes tools/devices used to passively provide the desired response. Pharmaceutical, complementary, and alternative medicines such as yoga, massage, and acupuncture will be excluded. There is no restriction on the intensity, duration, and provider of the intervention.\n\nThe outcomes of our scoping review include head and trunk control, gross motor function and activities of daily living (ADL). Head control can be defined as the capacity to maintain head alignment with body against gravity. Poor head control can be defined as inability to align head with reference to the body against gravity.27 Similarly, the ability to maintain trunk stable in sitting position for more than a minute and use upper extremity for functional movements is defined as trunk control. Poor trunk control can be defined as inability/difficulty to maintain the trunk stable in sitting position or requiring external support.9 Head and trunk control can be measured either qualitatively or with different tools or outcome measures. This scoping review will not impose any restriction on outcome measures.\n\nData file 2 in the Extended data illustrates conceptual framework of our research describing different physiotherapy interventions, their possible outcomes, and overall impact on children with developmental disabilities.\n\nContext: This scoping review will include studies conducted in a variety of settings, such as outpatient, inpatient, and community, country/region, urban, semi-urban, or rural settings. The scoping review will not have any geographic restrictions.\n\nTypes of sources: This review will include randomized controlled trials, non-randomized trials, quasi-experimental trials, and systematic reviews of interventional studies. The editorial, conference abstracts, letters, opinions, and comments will be excluded; however, if these employ the study designs, we will contact the corresponding authors and request to share the full papers to be included in the scoping review.\n\nInformation sources\n\nAll identified records will be collated and uploaded into Zotero, and duplicates will be removed. Rayyan QCRI (Qatar Computing Research Institute, Doha, Qatar), a web application, will be utilized to import unique citations, which permits screening, and administration of important screening statistics among reviewers.28 Two independent reviewers will be screening titles and abstracts against the reviewâs inclusion criteria. The full texts of chosen citations will be retrieved, and two independent reviewers will assess them against the eligibility criteria in detail. Reasons for excluding full-text articles that do not meet the inclusion criteria will be recorded and reported. At each stage of the selection process, any disagreements between the reviewers will be addressed by discussing with a third reviewer. PRISMA 2020 flow diagram will be utilized to present the screening results in the final scoping review.29\n\nSearch strategy\n\nA preliminary search of Medline (PubMed) and EMBASE was conducted on 18/06/2022 to identify appropriate keywords. A PubMed search strategy was constructed using identified keywords of relevant publications, data file 3. Furthermore, based on the finalized search strategy, the following databases will be searched: Medline (PubMed and Web of Science), Embase, the Cochrane Library, Scopus, CINAHL Complete, and PEDro. Additionally, we will search other sources (grey literature search and registries) such as, Australasian Academy of Cerebral Palsy and Developmental Medicine (AusACPDM), American Academy for Cerebral Palsy and Developmental Medicine (AACPDM), The WHO, CanChild Centre for Childhood Disability Research, ProQuest, MedNAR, OpenGrey, and clinicaltrial.gov. Furthermore, citation tracking through back referencing and forward citations of the included studies will be carried out. The search strategy will be modified according to the databases with the intention to carry out a comprehensive and up-to-date search. Articles published in English from the inception of databases to the date of the search will be included. Studies will be included irrespective of their publication status, irrespective of peer-reviewed or not, such as journal articles, reports, thesis, and dissertations are eligible for inclusion.\n\nData extraction\n\nData coding tool will be developed and used to code data from the papers included in the scoping review. Specific details regarding âparticipantsâ, âconceptâ, âcontextâ, âstudy methodsâ and âimportant findingsâ pertinent to the review questions will be included in the retrieved data. A data coding form will be designed and piloted based on the variables enlisted in Table 1. The ICF framework taxonomy will be utilized to determine the variables to be extracted from included studies. Additional or missing data (if any) from included studies will be obtained by contacting the authors of the articles. If the authors do not respond within two weeks of communication, the studies will be included, and appropriate limitations will be indicated. While reporting the interventions focused on head and trunk control in children with developmental disabilities, Consensus on Exercise Reporting Template (CERT)30 and Template for Intervention Description and Replication (TIDieR)31 will be used.\n\nData analysis and presentation\n\nWe will summarize objective 1 by preparing a list of interventions that were used for head and trunk control among children with developmental disabilities. We will summarize these interventions as per CERT and TIDieR. To achieve objective 2, we will map studies that assessed the effectiveness of intervention(s) on outcomes of our interest among children with developmental disabilities. We will group these studies based on study types e.g., RCTs, non-RCTs, and systematic reviews. Similarly, ongoing studies will also be documented.\n\nQualitative and quantitative findings of the studies will be tabulated. Visual representation (illustrations) will be utilized to highlight demographic details using frequency/percentage distribution of the included studies, details of intervention, type of study design, and country or location. We have preliminary mapped the evidence on physiotherapy and technology-based intervention for head and trunk control of children with developmental disabilities from one of the probably included studies using an example table (Table 2).\n\n\n\nâ Session: 3 times/day for 15 minutes (total of 45 minutes per day)\n\nâ Duration: 6 months\n\nâ Provider: Investigators trained parents to apply the headpods\n\n\n\n1) Head control\n\ni. Active time - the child held the head upright against gravity to participate in a motivating activity during a 5-minute video capture (3 & 6 months)\n\nii. Head bobs were counted using the same video captures (3 & 6 months)\n\nSecondary outcomes:\n\n2) Adherence as determined by daily recording of device use on a monthly log sheet (3 & 6 months)\n\n3) Perceived improvement via a 3-question, 15-point global rating of change (GROC) scale survey with an open-ended comment section (6 months)\n\nRisk of bias assessment, meta-analysis or Grading of Recommendations, Assessment, Development and Evaluations, is out of the scope of our scoping review.\n\nPatient and public involvement\n\nWe had an interaction with stakeholders (a parent and health professional), to get their feedback on defining the research topics. One of the stakeholders is a parent of a child with developmental disabilities, receiving regular therapy for the acquisition of head and trunk control. Another stakeholder is a pediatric physiotherapist who reviewed our protocol.\n\nEthics and dissemination\n\nWe will conduct a scoping review of existing studies and will not recruit participants directly; thus, ethical approval will not be required. We intend to present the results of the full scoping review at an international conference and submit the paper for publication in a peer-reviewed journal.\n\n\n\n⢠This scoping review will employ a comprehensive search in major databases and other sources (including grey literature search) to identify relevant studies.\n\n⢠Stakeholders (parent of child with developmental disability and health professional) were consulted to get feedback on this protocol.\n\n⢠Due to resource limitations, this review will be confined to publications in the English language.\n\nWe are currently identifying the keywords for search strategy and anticipate running the search in various databases at the end of September 2022.\n\n\nConclusion\n\nThis scoping review will emphasize the current research and knowledge gaps in physiotherapy and technology-based interventions for head and trunk control in children with developmental disabilities. Therefore, this information may be used by the scientific community to conduct interventional research or carry out a systematic review. Additionally, this can help funders prioritise research topics when they are seeking research proposal submissions.\n\n\nData availability\n\nNo data are associated with this article.\n\nOSF: Physiotherapy interventions for head and trunk control in children with developmental disabilities - A scoping review protocol. https://doi.org/10.17605/OSF.IO/B3RSU.26\n\nThis project contains the following extended data:\n\n- Data file 1 (Supplementary Table 1 listing the rehabilitation interventions used in physiotherapy for children with developmental disabilities)\n\n- Data file 2 (Appendix 1 is the figure illustrating the Conceptual Framework mapping the physiotherapy and technology-based interventions in children with Developmental Disabilities)\n\n- Data file 3 (Appendix 2 is the preliminary PubMed search strategy carried out on 18/06/2022)\n\n\nReporting guidelines\n\nOSF: PRISMA-P checklist for âPhysiotherapy interventions for head and trunk control in children with developmental disabilities - A scoping review protocolâ. https://doi.org/10.17605/OSF.IO/B3RSU.26\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)",
"appendix": "Acknowledgments\n\nWe are grateful to the stakeholders for providing us with their valuable feedback on defining the research topics. The author would like to acknowledge the Manipal Academy of Higher Education (MAHE), Manipal for technical support.\n\n\nReferences\n\nTecklin JS: Pediatric Physical Therapy. 5th ed.Baltimore, MD:Lippincott Williams & Wilkins;2015.\n\nOlusanya BO, Davis AC, Wertlieb D, et al.: Developmental disabilities among children younger than 5 years in 195 countries and territories, 1990â2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Glob. Health. 2018; 6: e1100âe1121. PubMed Abstract | Publisher Full Text\n\nDemirci A, Kartal M: The prevalence of developmental delay among children aged 3-60 months in Izmir, Turkey: Developmental delay among children. Child Care Health Dev. 2016; 42: 213â219. PubMed Abstract | Publisher Full Text\n\nStavsky M, Mor O, Mastrolia SA, et al.: Cerebral PalsyâTrends in Epidemiology and Recent Development in Prenatal Mechanisms of Disease, Treatment, and Prevention. Front. Pediatr. 2017; 5. PubMed Abstract | Publisher Full Text\n\nCaban-Holt A, Head E, Schmitt F:Down Syndrome. Rosenbergâs Molecular and Genetic Basis of Neurological and Psychiatric Disease. Elsevier;2015; 163â170. Publisher Full Text\n\nDangouloff T, Botty C, Beaudart C, et al.: Systematic literature review of the economic burden of spinal muscular atrophy and economic evaluations of treatments. Orphanet J. Rare Dis. 2021; 16: 47. PubMed Abstract | Publisher Full Text\n\nBrown JE, Thompson M, Brizzolara K: Head Control Changes After Headpod Use in Children With Poor Head Control: A Feasibility Study. Pediatr. Phys. Ther. 2018; 30: 142â148. PubMed Abstract | Publisher Full Text\n\nDusing SC, Izzo TA, Thacker LR, et al.: Postural complexity differs between infant born full term and preterm during the development of early behaviors. Early Hum. Dev. 2014; 90: 149â156. PubMed Abstract | Publisher Full Text\n\nPin TW, Butler PB, Cheung H-M, et al.: Relationship between segmental trunk control and gross motor development in typically developing infants aged from 4 to 12 months: a pilot study. BMC Pediatr. 2019; 19: 425. PubMed Abstract | Publisher Full Text\n\nSandlund M, Mcdonough S, HÀger-Ross C: Interactive computer play in rehabilitation of children with sensorimotor disorders: a systematic review. Dev. Med. Child Neurol. 2009; 51: 173â179. PubMed Abstract | Publisher Full Text\n\nVelasco MA, Raya R, Muzzioli L, et al.: Evaluation of cervical posture improvement of children with cerebral palsy after physical therapy based on head movements and serious games. Biomed EngOnLine. 2017; 16: 74. PubMed Abstract | Publisher Full Text\n\nInternational Classification of Functioning, Disability and Health (ICF): World Health Organ. Reference Source\n\nSoper AK, Cross A, Rosenbaum P, et al.: Knowledge translation strategies to support service providersâ implementation of the âF-words in Childhood Disability.â. DisabilRehabil. 2020; 43: 3168â3174. PubMed Abstract | Publisher Full Text\n\nHenderson S, Skelton H, Rosenbaum P: Assistive devices for children with functional impairments: impact on child and caregiver function. Dev. Med. Child Neurol. 2008; 50: 89â98. PubMed Abstract | Publisher Full Text\n\nRyan JM, Cassidy EE, Noorduyn SG, et al.: Exercise interventions for cerebral palsy. Cochrane Database Syst. Rev. 2017; 2017: CD011660. PubMed Abstract | Publisher Full Text\n\nKerem M, Turker D, Ozal C, et al.:Physical Management of Children with Cerebral Palsy.Vraka E, editor. Cerebral Palsy - Challenges for the Future. InTech;2014. Publisher Full Text\n\nFurtado MAS, Ayupe KMA, Christovão IS, et al.: Physical therapy in children with cerebral palsy in Brazil: a scoping review. Dev. Med. Child Neurol. 2021; 64: 550â560. PubMed Abstract | Publisher Full Text\n\nKumar R, Prakash A: Efficacy of cervical orthoses in improving neck control of growing children with cerebral palsy: A narrative review. J Indira Gandhi Inst Med Sci. 2021; 7: 90. Publisher Full Text\n\nWorld Health Organization: World health statistics 2018: monitoring health for the SDGs, sustainable development goals. Geneva:World Health Organization;2018. (accessed 17 Nov 2021).Reference Source\n\nUnited Nationâs Sustainable Developmental Goals and Disability:(accessed 25 Jun 2022).Reference Source\n\nJindal P, Macdermid JC, Rosenbaum P, et al.: Treatment and re/habilitation of children with cerebral palsy in India: a scoping review. Dev. Med. Child Neurol. 2019; 61: 1050â1060. PubMed Abstract | Publisher Full Text\n\nSantamaria V, Khan M, Luna T, et al.: Promoting Functional and Independent Sitting in Children With Cerebral Palsy Using the Robotic Trunk Support Trainer. IEEE Trans Neural Syst RehabilEng. 2020; 28: 2995â3004. PubMed Abstract | Publisher Full Text\n\nSibley KM, Beauchamp MK, Van Ooteghem K, et al.: Components of Standing Postural Control Evaluated in Pediatric Balance Measures: A Scoping Review. Arch. Phys. Med. Rehabil. 2017; 98: 2066â2078.e4. PubMed Abstract | Publisher Full Text\n\nDoctor KN, Karnad SD, Krishnan SK, et al.: Understanding Postural and Segmental Trunk Control and Their Effect on Sitting in Children with Cerebral Palsy: A Systematic Scoping Review. Crit Rev Phys Rehabil Med. 2021; 33: 25â45. Publisher Full Text\n\nPeters M, Godfrey C, McInerney P, et al.:Chapter 11: Scoping Reviews.Aromataris E, Munn Z, editors. JBI Manual for Evidence Synthesis. JBI;2020. Publisher Full Text\n\nShakya S, Parsekar S, Ramachandran S, et al.: Physiotherapy interventions for head and trunk control in children with developmental disabilities - A scoping review protocol.Published Online First: 22 August 2022. Publisher Full Text\n\nleckey_clinical_head_control_in_children_with_cp.pdf:(accessed 6 Jan 2022).Reference Source\n\nOuzzani M, Hammady H, Fedorowicz Z, et al.: Rayyanâa web and mobile app for systematic reviews. Syst. Rev. 2016; 5: 210. PubMed Abstract | Publisher Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. Int. J. Surg. 2021; 88: 105906. PubMed Abstract | Publisher Full Text\n\nSlade SC, Dionne CE, Underwood M, et al.: Consensus on Exercise Reporting Template (CERT): Explanation and Elaboration Statement. Br. J. Sports Med. 2016; 50: 1428â1437. PubMed Abstract | Publisher Full Text\n\nHoffmann TC, Glasziou PP, Boutron I, et al.: Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ. 2014; 348: g1687. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "161522",
"date": "14 Feb 2023",
"name": "Jodi Thomas",
"expertise": [
"Reviewer Expertise Pediatric Physical Therapy-development and validation of a scale to assess head control"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article outlines the planned protocol for this scoping review well. The steps that will be taken are defined and clear. This scoping review aims to find studies regarding physiotherapy and technology based interventions that are used in the treatment of head and neck control deficits. The authors mention the need to address ICF components such as activity limitations; I think it would be beneficial for the data extraction to include how the intervention addressed ICF components. One item that is unclear to me is why the age range is set at 2 years, excluding articles that include children younger than 2 years. The rationale for this was not made clear. Overall, this protocol demonstrates sound scientific design and should results in information that is useful to the physiotherapy community as well as many others.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "9450",
"date": "12 Apr 2023",
"name": "Shristi Shakya",
"role": "Author Response",
"response": "Thank you so much for your valuable suggestions and feedback. We are grateful to you for sparing your valuable time to review our manuscript. We have addressed your valuable comments/suggestion in the revised manuscripts along with some of our minor revisions. Q: The authors mention the need to address ICF components such as activity limitations; I think it would be beneficial for the data extraction to include how the intervention addressed ICF components. A: Thank you for your comments/suggestions. We have implemented the ICF taxonomy in the âPreliminary data coding variablesâ segment, in the updated version of the manuscript. Q: One item that is unclear to me is why the age range is set at 2 years, excluding articles that include children younger than 2 years. The rationale for this was not made clear. A: Thank you so much for your valuable comment. Yes, it is a valid point as head control is acquired at the age of 4 to 6 months. Therefore, we have incorporated the changes and have modified the age group to 6 months to 17 years 11 months and 29 days, in the âParticipantsâ segment of the Eligibility Criteria in the manuscript."
}
]
},
{
"id": "162768",
"date": "28 Feb 2023",
"name": "Emily C. Marcinowski",
"expertise": [
"Reviewer Expertise motor development",
"infancy",
"active play",
"motor delays or disorders",
"cultural variations in dyadic play and development"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, I found this review protocol to be organized well and clear with its goals. Iâve include a few comments below that I believe will improve it further:\nMethods:\n(Table 1/3. Geographical Information): I would consider including information about a communityâs resources for providing intervention to these populations (e.g., health care structure, access to Early Intervention). By providing this, readers can see the standard of care of a studyâs region and potentially describe accessibility and efficacy of a technology-based intervention.\n(Protocol / Eligibility criteria): âFurthermore, this review will exclude population having acquired conditions affecting head and trunk control.â Can you give some examples of what these would include?\n(Protocol / Patient and public involvement): Please add a brief description of what the stakeholders provided to the review. Information on scope of the review? Terminology? Labor? Priorities?\nConclusion:\nYou primarily discuss this review is to fill in the gaps to improve interventional research and prioritize funding goals. I think it makes sense to describe more fully how these findings lend themselves to quality, evidence-based practices for technology-based interventions. Technology is unique in that it can improve or limit accessibility to the community, depending on its cost, necessary equipment, and location/community resources available. By widening your discussion points to include this point, I think it would widen your audience and make a better point for researchers and funders to consider what is necessary for technology-based interventions.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "9451",
"date": "12 Apr 2023",
"name": "Shristi Shakya",
"role": "Author Response",
"response": "Thank you so much for your valuable comments/suggestions which provided us lots of insights to strengthen our protocol even further. We have addressed your valuable comments/suggestion in the revised manuscripts along with some of our minor revisions and is track changed. Methods: Q: (Table 1/3. Geographical Information): I would consider including information about a communityâs resources for providing intervention to these populations (e.g., health care structure, access to Early Intervention). By providing this, readers can see the standard of care of a studyâs region and potentially describe accessibility and efficacy of a technology-based intervention. A: Thank you so much for your suggestions. We have included the information in the âcontextâ segment of the Eligibility Criteria of the manuscript and âgeographical locationâ segment in the preliminary data coding variables. Q: (Protocol / Eligibility criteria): âFurthermore, this review will exclude population having acquired conditions affecting head and trunk control.â Can you give some examples of what these would include? A: This review will include children and adolescents from 6 months to less than 18 years of age with developmental disability including Down Syndrome, Spinal Muscular Atrophy, Developmental Delay and Cerebral Palsy, having poor head and trunk control. Head control can be defined as the capacity to maintain head alignment with body against gravity. Poor head control can be defined as inability to align head with reference to the body against gravity. Additionally, the ability to maintain trunk stable in sitting position for more than a minute and use upper extremity for functional movements is defined as trunk control. Poor trunk control can be defined as inability/difficulty to maintain the trunk stable in sitting position or requiring external support. Q: (Protocol / Patient and public involvement): Please add a brief description of what the stakeholders provided to the review. Information on scope of the review? Terminology? Labor? Priorities? A: The stakeholders (a parent and health professional), have provided us with their valuable feedback regarding the necessity of conducting this review and on defining the research topics. One of the stakeholders is a parent of a child with developmental disabilities, receiving regular therapy for the acquisition of head and trunk control. Another stakeholder is a pediatric physiotherapist who reviewed our protocol. Q: You primarily discuss this review is to fill in the gaps to improve interventional research and prioritize funding goals. I think it makes sense to describe more fully how these findings lend themselves to quality, evidence-based practices for technology-based interventions. Technology is unique in that it can improve or limit accessibility to the community, depending on its cost, necessary equipment, and location/community resources available. By widening your discussion points to include this point, I think it would widen your audience and make a better point for researchers and funders to consider what is necessary for technology-based interventions. A: Thank you for your valuable comments. We have incorporated these modifications in the âReview Questionâ and âconclusionâ segments of the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1074
|
https://f1000research.com/articles/12-294/v1
|
16 Mar 23
|
{
"type": "Genome Note",
"title": "The complete mitochondrial genome Hainan Gymnure Neohylomys hainanensis",
"authors": [
"Feiyun Tu",
"Xiaodan Hou",
"Qiujie Lu",
"Xiaofei Zhai",
"Xiaodan Hou",
"Qiujie Lu",
"Xiaofei Zhai"
],
"abstract": "The Hainan Gymnure Neohylomys hainanensis is a small-size mammal which occurs in Hainan, China and north Vietnam. Here, we report the complete mitochondrial genome of N. hainanensis. The whole mitochondrial genome is 17,337 bp, and contains 13 protein-coding genes, 22 tRNAs, 2 rRNAs and one control region. The base composition of the N. hainanensis total mitogenome is: 33.4% A, 12.2% G, 33.1% T, and C 21.3%, with an A + T content of 66.5%. The K2P genetic distance analysis supports current taxonomy that places the hainanensis, sinensis and suillus in different genera. Phylogenetic analysis suggests that N. hainanensis is closely related to Neotetracus sinensis based on the complete mitochondrial genome sequences. The mitogenomic data will contribute to molecular phylogenetics and conservation genetics of the species.",
"keywords": [
"Neohylomys hainanensis",
"mitochondrial genome",
"phylogeny",
"Erinaceidae"
],
"content": "Introduction\n\nThe Hainan Gymnure Neohylomys hainanensis Shaw and Wang, 1959 is mainly distributed in Hainan, China and north Vietnam (Liu and Wu 2019; Abramov et al. 2018). It mainly inhabits the tropical forests and subtropical forests (Smith et al. 2009). To date, the phylogenetic relationships of three species (N. hainanensis, Hylomys suillus and Neotetracus sinensis) within Erinaceidae have been debated (Cobert 1988; Gould 1995; Grenyer and Purvis 2003; He et al. 2012). Previous studies (Cobert 1988; Gould 1995; Grenyer and Purvis 2003) indicated sister relationships of N. hainanensis and N. sinensis based on morphological data. However, He et al. (2012) showed that N. sinensis clustered with H. suillus rather than N. hainanensis based on combined data of mitochondrial genes (CYTB, ND2 and 12S). Complete mitochondrial genome sequences are highly informative markers for resolving high level phylogenetic relationships among taxa (Kumazawa 2007; Shen et al. 2010; Yue et al. 2015). Though the complete mitochondrial genome of N. hainanensis is available in GenBank (MW429379), we sequenced the mitochondrial genome using our sample based on primer walking method to confirm the accuracy of the data. Additionally, we investigated phylogenetic relationships of N. hainanensis among family Erinaceidae to solve the disputes.\n\n\nMethods\n\nThe animal was captured using a mouse cage trap at Yinggeling Mountains (109.289°E, 18.878°N), Hainan province, China in May, 2022. The animal was euthanized in the euthanasia cage (10L), which was gradually filled with 99% purity of CO2 within 2-3 minutes.\n\nA total of 2 g of femoral muscle sample was clipped from the specimen using surgical scissors, and the total genomic DNA was extracted from the muscle tissue using the phenolâchloroform extraction (Sambrook et al. 1989), which contained three main procedures: 1) Sodium dodecylsulfate (SDS) and proteinase K were used for the enzymatic digestion of proteins; 2) A mixture of phenol: chloroform:isoamyl alcohol (25:24:1) was then added to promote the partitioning of lipids into the organic phase; 3) The DNA was rinsed using analytical alcohol of different solubility and then otained the purified DNA for PCR. Seven primer combinations were used for the generation of PCR products (see Extended data (Tu 2023)). PCR amplifications of mitochondrial genome of N. hainanensis were listed as follows: PCR amplifications were carried out in 25 uL reaction volumes containing 1ÃEX Taq buffer (Mg2+ Free; TaKaRaBiotech, Dalian, China), 2.5 mM MgCl2, 0.1-0.4 mM dNTP, 0.4 uM each primer, 0.04 u/uL EX Taq polymerase, and ~5 ng/uL total genomic DNA as template. PCR protocol was set as follows: an initial denaturation at 94°C for 4 min; 35 cycles of amplification as the main procedure: a denaturation at 94°C for 45 s, an annealing at 50-60°C for 50 s, an elongation at 72°C for 2-3 min, and a final extension at 72°C for 4 min. All PCR products were examined through electrophoresing on a 1.0% agarose gel and then directly sequenced using an ABI 3730xl sequencer. All sequences were assembled and edited using SeqMan (DNASTAR 7.1.0) (Swindell and Plasterer 1997). The boundaries of protein-coding genes were predicted via comparison with homologs using MEGA 6.0 (Tamura et al. 2013). The transfer RNA (tRNA) genes were identified using tRNAscan-SE 2.0 (Lowe and Chan 2016).\n\nTo better understand the phylogentic position of N. hainanensis within Eulipotyphla, we constructed a phylogenetic tree by using neighbor-joining (NJ) method implemented in MEGA6.0 (Tamura et al. 2013) based on 13 complete mitochondrial genome sequences of Eulipotyphla using Kimura 2-parameters (K2P) model.\n\n\nResults\n\nThe whole mitochondrial genome of N. hainanensis is 17,337 bp in length (GenBank Accession number ON054206.2 (Tu and Hou 2022)) and contains 13 protein-coding genes (PCGs), 22 tRNAs genes, two rRNAs genes, and one control region. The base composition of N. hainanensis mitogenome is as follows: A 33.4%, G 12.2%, T 33.1%, and C 21.3%. Typically, an A+T rich pattern (66.5%) was observed. Comparison with a previously published mitochondrial genome sequence (MW429379) for the same species, these two sequences differ in length (17,337 bp vs 16,795 bp), which may be due to mitochondrial control region sequence variations (1,868 bp vs 1334bp). Within 37 mitochodrial genes, the ND6 gene and eight tRNAs (tRNA Gln, tRNA Ala, tRNA Asn, tRNA Cys, tRNA Tyr, tRNA Ser, tRNA Glu and tRNA Pro) were encoded on the L-strand, whereas all other genes were encoded on the H-strand. Most mitochondrial PCGs, use ATG as its start codon, ND2 and ND3 begin with ATT, and ND5 begin with ATA. Five genes (COX2, ATP6, ND3, ND4L and ND5) use TAA as stop codons, whereas four genes (ND1, ND2, COX1 and ATP8) end with TAG, and ND6 ends with AGA. COX3, ND4 and CYTB use T as an incomplete stop codon.\n\nA phylogenetic tree showed that two sequences of N. hainanensis shared a common evolutionary ancestry. Within subfamily Galericinae, H. suillus and the common ancestors of N. hainanensis and N. sinensis shaped sister groups (Figure 1). The results were consistent with morphological studies (Cobert 1988; Gould 1995; Grenyer and Purvis 2003). Our results support current taxonomy that places the hainanensis, sinensis and suillus in different genera (Smith et al. 2009; Wei et al. 2021). This mitochondrial genome sequence determined in this study will benefit conservation genetics and phylogenetics of the species in future.\n\nNodal support indicated by bootstrap.\n\nThis work complies with the International Union for Conservation of Nature (IUCN) policies research involving species at risk of extinction (see Guidelines for appropriate uses of IUCN Red list data version 4.0; https://www.iucnredlist.org/resources/guidelines-for-appropriate-uses-of-red-list-data), as the species under study is an endangered species. All procedures were approved by Animal Research Ethics Committee of Hainan Provincial Education Center for Ecology and Environment, Hainan Normal University (HNECEE-2022-003) on March 6, 2022.",
"appendix": "Data availability\n\nNCBI Nucleotide: Neohylomys hainanensis voucher HN20N051 mitochondrion, complete genome. Accession number ON054206.2, https://www.ncbi.nlm.nih.gov/nuccore/ON054206.2 (Tu and Hou 2022).\n\nFigshare: Neohylomys hainanensis primer combinations data. https://doi.org/10.6084/m9.figshare.22180219.v1 (Tu 2023).\n\nThis project contains the following extended data:\n\n- Primer information\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAbramov AV, Bannikova AA, Lebedev VS, et al.: A broadly distributed species instead of an insular endemic? A new find of the poorly known Hainan gymnure (Mammalia, Lipotyphla). ZooKeys. 2018; 795: 77â81. Publisher Full Text\n\nCobert GB: The family Erinaceidae: a synthesis of its taxonomy, phylogeny, ecology and zoogeography. Mammal Rev. 1988; 18(3): 117â172.\n\nGould GC: Hedgehog phylogeny (Mammalia, Erinaceidae): The reciprocal illumination of the quick and the dead. Am. Mus. Novit. 1995; 1â45.\n\nGrenyer R, Purvis A: A composite species-level phylogeny of the âInsectivoraâ (Mammalia: Order Lipotyphla Haeckel,1866). J. Zool. 2003; 260: 245â257. Publisher Full Text\n\nHe K, Chen JH, Gould GC, et al.: An Estimation of Erinaceidae Phylogeny: A Combined Analysis Approach. PLoS One. 2012; 7(6): e39304. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKumazawa Y: Mitochondrial genomes from major lizard families suggest their phylogenetic relationships and ancient radiations. Gene. 2007; 388: 19â26. PubMed Abstract | Publisher Full Text\n\nLiu SY, Wu Y: Handbook of the mammals of China. Fuzhou: The Strait Publishing & Distributing Group; 2019.\n\nLowe TM, Chan PP: tRNAscan-SE On-line: search and contextual analysis of transfer RNA genes. Nucleic Acids Res. 2016; 44(W1): W54âW57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShen YY, Liang L, Sun YB, et al.: A mitogenomic perspective on the ancient, rapid radiation in the Galliformes with an emphasis on the Phasianidae. BMC Evol. Biol. 2010; 10: 132. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith AT, Xie Y, Wang S: A Guide to the Mammals of China. Changsha: Hunan Education Press; 2009.\n\nTamura K, Stecher G, Peterson D, et al.: MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 2013; 30: 2725â2729. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTu F: Primer information.docx. Dataset. figshare. 2023. Publisher Full Text\n\nTu F, Hou X: Neohylomys hainanensis voucher HN20N051 mitochondrion, complete genome. [Dataset]. NCBI GenBank. 2022. Reference Source\n\nWei FW, Yang QS, Wu Y, et al.: Catalogue of mammals in China. Acta Theriologica Sinica. 2021; 41(5): 487â501.\n\nYue H, Yan CC, Tu FY, et al.: Two novel mitogenomes of Dipodidae species and phylogeny of Rodentia inferred from the complete mitogenomes. Biochem. Syst. Ecol. 2015; 60: 123â130. Publisher Full Text"
}
|
[
{
"id": "255823",
"date": "04 Apr 2024",
"name": "Michael Allen",
"expertise": [
"Reviewer Expertise Conservation",
"Environmental DNA",
"DNA metabarcoding",
"Bioinformatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments\nThis is a useful study in that it provides new genetic information for a poorly studied endangered species, the mammal Neohylomys hainanensis. The mitochondrial genome described therein will prove useful for future phylogenetic research as well as for metabarcoding studies of distribution and habitat use. While there was already a single complete genome for the species available in GenBank, the new genome and additional analysis provided by the authors provides novel information regarding intra- and inter-specific variation within the species and clade. The value of the paper could be enhanced to the extent that the authors can expand and elaborate on these analyses.\nDetailed comments\nTITLE\nSuggest changing the title to the following to improve clarity: âThe complete mitochondrial genome of the Hainan Gymnure, Neohylomys hainanensisâ\nABSTRACT\nââŠthe hainanensis, sinensis and suillusâŠâ: These three species should be identified by their full binomial name here, not just the specific epithet. E.g., âN. hainanensis, Hylomys suillus and Neotetracus sinensisâ\nIt would be good to report more detail here about what type of phylogenetic analysis was performed, if possible.\nINTRODUCTION\nParagraph 1, sentences 1 & 2: Suggest rephrasing as follows: âThe Hainan Gymnure (Neohylomys hainanensis Shaw and Wang, 1959) is a rodent distributed in Hainan, China and north Vietnam, where it inhabits tropical and subtropical forests (Smith et al. 2009; Liu and Wu 2019; Abramov et al. 2018).â I believe this would improve the flow and would serve to inform the reader what type of organism it is (i.e., a Mammal and a Rodent), information which is not currently provided.\nPlease state how many species are currently recognized within the family Erinaceidae.\nParagraph 1, sentence 6: suggest deleting the words âhigh levelâ as their meaning is unclear here.\nParagraph 1, sentence 8: suggest changing ââŠto solve the disputes.â to ââŠin an attempt to resolve the current phylogenetic disputes.â\nMETHODS\nMethods paragraph 1, sentence 2: I recommend changing ââŠin the euthanasia cage (10L)âŠâ to ââŠin a 10 L euthanasia cageâŠâ. And, if possible, describe the cage and its material as the phrase âeuthanasia cageâ is not self-evident.\nMethods paragraph 2, sentence 1: delete the word âtheâ from the phrase âusing the phenolâchloroform extractionâ.\nMethods paragraph 2, âAll sequences were assembled and edited using SeqMan (DNASTAR 7.1.0) (Swindell and Plasterer 1997).â: Surely there must be more methods to report here in addition to which software you used. Are there any more details about settings used, etc., you can provide that would help someone better understand and repeat this analysis?\nMethods paragraph 3: âMEGA6.0â should have a space in it.\nMethods paragraph 3 (â13 complete mitochondrial genome sequences of Eulipotyphlaâ): Please either state which species genomes were used here, or else reference Table 1 which I think provides that information. Also, please address why are there only 11 genomes displayed in Table 1 given that you analyzed 13. It would also be good to report how many species and genera are currently known to occur within Eulipotyphla. That would provide readers a better understanding of how complete your analysis is.\nRESULTS\nResults paragraph 1, sentence 4: sentence is incomplete as written. Please change to âComparison with a previously published mitochondrial genome sequence (MW429379) for the same species revealed that these two sequences differ in length (17,337 bp vs 16,795 bp), which may be due to mitochondrial control region sequence variations (1,868 bp vs 1334bp).â\nResults paragraph 1, penultimate sentence: Contains minor punctuation and wording issues. Rephrase as follows: âMost mitochondrial PCGs contain ATG as the start codon, ND2 and ND3 begin with ATT, and ND5 begins with ATA.â\nResults paragraph 2, sentence 2: The word âshapedâ should be replaced with the word âformedâ to improve clarity.\nResults paragraph 2, sentence 4: Please include the genus names or initials for these specific epithets. (See comment in Abstract.)\nResults paragraph 2, last sentence: Change ending of sentence as follows to improve clarity: ââŠwill benefit future research into conservation genetics and phylogenetics for the species.â\nIt would be useful to include summary statistics regarding pairwise K2P differences for different regions (e.g., in the context of DNA barcoding and metabarcoding).\nDISCUSSION\nConsider including a brief Discussion section if permitted by the journal format.\nFIGURES\nFigure 1: Please include in the caption the fact that this represents 10 species from the Eulipotyphla clade. Also, briefly explain that they were created using a neighbor-joining tree and that the codes after the species names represent GenBank accession numbers (if true).\nTABLES\nWhile not required, a table comparing the various attributes of the 2 known genomes of N. hainanensis (and perhaps other species analyzed) would be welcome.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
},
{
"id": "250944",
"date": "11 May 2024",
"name": "Perinçek Seçkinozan Åeker",
"expertise": [
"Reviewer Expertise Molecular Systematics Zoology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report the complete mitochondrial DNA sequence of an insectivorous mammal species whose complete mitochondrial genome is already available at NCBI to confirm the accuracy of the data https://www.ncbi.nlm.nih.gov/nuccore/MW429379. At this point, the logic of sequencing the genome of this species and the importance of the species, which are the main issues that will emphasize the importance of the article, have not been sufficiently justified. Although this is even a genome announcement, this section should be developed by clearly stating the rationales. The methods used are up-to-date and fit for the study. The obtained genome sequence has been presented in a usable and accessible format in NCBI. A previously reported genome sequence of this species is available at NCBI. Therefore, it would be better to give the results of statistical analyses (for example, the topology of the phylogenetic tree and K2P distances) and the structures of the genomes comparatively. For example, there is a difference in length between the two genomes Why does this situation occur? It should be presented by comparing. As a result, this study presented as a genome note can be indexed if these requirements are improved.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-294
|
https://f1000research.com/articles/12-287/v1
|
15 Mar 23
|
{
"type": "Review",
"title": "Progress and challenges for the application of machine learning for neglected tropical diseases",
"authors": [
"ChungYuen Khew",
"Rahmad Akbar",
"Norfarhan Mohd-Assaad",
"ChungYuen Khew"
],
"abstract": "Neglected tropical diseases (NTDs) continue to affect the livelihood of individuals in countries in the Southeast Asia and Western Pacific region. These diseases have been long existing and have caused devastating health problems and economic decline to people in low- and middle-income (developing) countries. An estimated 1.7 billion of the worldâs population suffer one or more NTDs annually, this puts approximately one in five individuals at risk for NTDs. In addition to health and social impact, NTDs inflict significant financial burden to patients, close relatives, and are responsible for billions of dollars lost in revenue from reduced labor productivity in developing countries alone. There is an urgent need to better improve the control and eradication or elimination efforts towards NTDs. This can be achieved by utilizing machine learning tools to better the surveillance, prediction and detection program, and combat NTDs through the discovery of new therapeutics against these pathogens. This review surveys the current application of machine learning tools for NTDs and the challenges to elevate the state-of-the-art of NTDs surveillance, management, and treatment.",
"keywords": [
"Neglected Tropical Diseases",
"Machine Learning",
"Drug Development",
"Drug Discovery."
],
"content": "Introduction\n\nCommunicable diseases are illnesses brought on by pathogens such as bacteria, viruses, parasitic worms, and fungi that can be contracted easily from contaminated surfaces, water, air droplets, air, bites from vector organisms, and direct contact with infected individuals. A recent Coronavirus disease (COVID-19) pandemic caused by the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), with 623,470,447 confirmed cases of COVID-19, and 6,551,678 reported deaths, as of 20th October 2022 (https://covid19.who.int/), underlines the need for highly efficient approaches to manage, surveil, and develop new treatments for communicable diseases. Unlike the COVID-19 pandemic, a âsilent yet horrific endemicâ caused by a diverse group of diseases affecting more than 149 countries and with more than 1.7 billion of infected individuals worldwide does not receive the same amount of attention (Jannin et al. 2017). The diverse group of diseases was coined by Peter Hotez and colleagues as âneglected tropical diseasesâ (NTDs) as they do not receive the same urgency, in neither treatments nor surveillance, as other diseases such as AIDS, malaria, and tuberculosis (Winkler et al. 2018). The World Health Organization (WHO) first established a list of 17 âofficialâ NTDs (World Health Organization 2010). Later in 2017, an addition of 3 disease conditions were made at the end of the 10th meeting of the Strategic and Technical Advisory Group for Neglected Tropical Diseases (World Health Organization 2017). 20 NTDs recognized by WHO are shown in Table 1.\n\n* Newly added diseases conditions into the NTD list prior to the outcome of the 10th meeting of the Strategic and Technical Advisory Group for Neglected Tropical Diseases.\n\nHigh incidences of NTDs were commonly reported from tropical countries due to its optimal humidity and climate for the pathogens to thrive. African and Asian countries with low and moderate incomes tend to lack proper access to clean water and waste management, thus greatly contributing to the spread of NTDs among women and children. To measure the extent of devastation caused by NTDs, the disability-adjusted life year (DALY; one DALY represents the loss of the equivalent of one year of full health) metric was introduced as a means to quantify the overall burden of disease borne by individuals (Mitra & Mawson 2017). DALYs for a disease or health condition are the sum of years of life lost (YLLs) due to premature mortality and years of healthy life lost due to disability (YLDs) due to prevalent cases of the disease or health condition in a population (Vinkeles Melchers et al. 2021). Based on the data collected by WHO, we were able to summarize the global burden for 14 of the 20 NTDs as estimated by DALYs in Table 2 below. Global burden for five of the highest estimated DALY burden NTDs are rabies (2.635 million years), dengue (1.952 million years), soil-transmitted helminthiases (STHs) (1.943 million years), schistosomiasis (1.628 million years), and lymphatic filariasis (LF) (1.616 million years). In another report from the Global Burden of Disease Study 2019, the estimated DALYs of NTDs was 15.142 million years, with the highest burden for dengue, followed by STHs, schistosomiasis, LF, and cysticercosis (Vos et al. 2020). When grouped based on category, helminth infections are responsible for the highest DALYs burden and widespread debilitating illnesses, with STHs as the leading cause of human helminthiases (Vos et al. 2020; World Health Organization 2020).\n\n\n\na) Ascariasis\n\n\n\nb) Trichuriasis\n\n\n\nc) Hookworm disease\n\na World Health Organization (2020).\n\nb Vos et al. (2020).\n\nTo grasp the disproportionate burden of NTDs worldwide, we summarized the DALY for each WHO region in Table 3. Based on the DALYs estimate by cause and WHO region in 2019, dengue has the highest burden of 1.510 million DALYs, followed by LF (1.029 million years), STHs (0.616 million years), rabies (0.455 million years), and cysticercosis (0.109 million years) in WHO-SEA Region.\n\n\n\na) Ascariasis\n\n\n\nb) Trichuriasis\n\n\n\nc) Hookworm disease\n\nDengue fever is a mosquito-borne viral disease spread to humans by the bites of infected female mosquitoes, primarily of the Aedes aegypti species (Simo et al. 2019). The disease is caused by members of the genus Flavivirus, within the Flaviviridae family (Molyneux 2019). Members of Flavivirus are responsible for a wide range of other deadly infections, whereby the virus that causes dengue comprises four different but closely related serotypes: DENV-1, DENV-2, DENV-3 and DENV-4 (Braack et al. 2018). These viruses are capable of causing illnesses of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) (Wibawa & Satoto 2016). In the past two decades, a 10-fold increase of 505,430 cases in 2000 to more than 5.2 million dengue cases were observed in 2019 (World Health Organization 2021). GBD 2019 estimated 2.38 million DALYs lost and age-standardized rates of 32.1 DALYs per 100,000 (95% UI 11.1 â 44.1) (Vos et al. 2020).\n\nLymphatic filariasis (LF) is caused by a group of helminths (roundworm) from the family of Filariodidea that reside in the lymphatic systems of humans (Wibawa & Satoto 2016). Wuchereria bancrofti is the primary infectious agent globally, with Brugia malayi and Brugia timori as runner-ups for LF infections (Mitra & Mawson 2017). Mosquitoes from the genera of Anopheles, Culex, Aedes, and Mansonia are responsible for the spread of LF (Famakinde 2018). Like many mosquito-borne diseases, mosquitoes picked up microfilariae when feeding on an infected LF individual. Then, the parasite develops within the mosquito and subsequently infects the next victim on the subsequent blood meal (Douglass et al. 2017). The devastating harm of having adult parasite nests and microfilaria discharged into the circulation of a person's lymphatic vessels leads to disease morbidity. Functionally impaired lymphatic systems lead to the manifestation of lymphoedema (elephantiasis), hence the enlarged state of the patient's limbs. Physically impaired people experience years of disability, stigmatization, and mental health comorbidity (Abela-Ridder et al. 2020). GBD 2019 estimated a DALYs burden of 1.630 million years, with age-standardized rates of 20.7 DALYs per 100,000 (95% UI 12.2 â 34.7) (Vos et al. 2020).\n\nSoil-transmitted helminths (STHs) typically infect the hostâs gastrointestinal region. WHO focuses on three STHs illnesses namely ascariasis, trichuriasis, and hookworm diseases. Ascariasis and trichuriasis are spread through ingestion of fecal-contaminated food or water (Muñoz-Antoli et al. 2022). Hookworm infection can be contracted when going barefoot on contaminated ground, in which the larvae develop into a form that enables them to pierce through human skin (Bethony et al. 2006). Studies revealed that the presence of high prevalence of STH are due to poor sociodemographic and socioeconomic status, especially in rural areas with poor infrastructure facilities, improper sewage and waste management, inadequate water supply, prolonged direct contact with soil such as walking barefooted, and poor sanitation and self-hygiene (Alelign, Degarege & Erko 2015; Ali et al. 2020). GBD 2019 estimated similar figures to WHO GHE 2019, with DALYs burden of 1.970 million years and age-standardized rates of 26.6 DALYs per 100,000 (95% UI 17 â 40.5) (Vos et al. 2020).\n\nRabies is a zoonotic disease from the Lyssavirus genus in the Rhabdoviridae family that has the capability to infect all mammalian lifeforms (Condori et al. 2020). The disease is a lyssavirus-induced acute, progressive encephalitis and has caused a high count of human mortality and economic consequences (World Organisation for Animal Health 2008; Brown et al. 2016). Once infected, the disease often results in inflammation of the brain's active tissues, causing headaches, stiff necks, light sensitivity, mental confusion, and seizures (Simon et al. 2013). Bite wounds or contamination of open cut wounds with infected saliva are primarily how the virus is transmitted (World Organisation for Animal Health 2008). Individuals in close contact with any suspected rabid animals should seek for post-exposure prophylaxis (PEP) treatment as a necessary means to prevent human rabies infection (Bamaiyi 2015). The virus proliferates on the central nervous system (CNS) and thus laboratory techniques on sample processing are focused at said area (Sataloff, Johns & Kost 2018). GBD 2019 estimated a DALYs burden of 782,000 years with age-standardized rates of 10.6 DALYs per 100,000 (95% UI 4.4 â 14.7) (Vos et al. 2020).\n\nCysticercosis is a cestode infection in both humans and porcine, caused by parasitic larval form (cysticercus) of Taenia solium, after consuming food or water contaminated with feces containing T. solium eggs (fecal-oral contamination) (Lustigman et al. 2012). Tapeworm eggs are spread when the carrier defecates in open areas (WHO 2014). When the eggs are consumed, they hatch in the colon, releasing oncospheres that breach the intestinal wall and enter the bloodstream. From there, they migrate to various tissues and organs (including the muscles, skin, eyes, and central nervous system), where they develop into cysticerci (Galipó et al. 2021). Cysticercosis can still develop in communities that do not consume pork or share habitats with pigs since this disease is spread by swallowing T. solium eggs that are shed in the feces of a human T. solium carrier. Development of parasitic cysts in the brain or central nervous system is referred to as neurocysticercosis (NCC) (Laranjo-González et al. 2017). GBD 2019 estimated a higher DALYs burden of 1.370 million years with age-standardized rates of 16.8 DALYs per 100,000 (95% UI 10.7 â 23.9) (Vos et al. 2020).\n\nMelioidosis or Whitmoreâs disease is a bacterial infection caused by Burkholderia pseudomallei, a soil- and water-borne Gram-negative bacterium capable of causing illness ranging from an acute or chronic localized infection to a widespread septicemic infection in multiple organs (Cheng & Currie 2005). Cases of melioidosis are frequently reported in endemic countries such as Africa, Australia, China India, Middle East, and Southeast Asia (typically Malaysia, Singapore, and Thailand) (Cheng & Currie 2005; Galyov, Brett & Deshazer 2010). Since its discovery in 1912, the bacteria still remains a topic of discussion among researchers due to its zoonotic nature, limited therapeutic options with no available vaccines till date (Borlee et al. 2017). Moreover, B. pseudomallei was designated as a Tier 1 select agent given its biothreat potential including high morbidity and mortality rates in low infectious doses, multidrug antibiotic resistance, and the amenability to be aerosolized (Hatcher, Muruato & Torres 2015). Melioidosis infection can be acquired through many routes with skin inoculation and inhalation or ingestion of contaminated water and air droplets to be the leading cause (Larsen & Johnson 2009). The disease mimics the signs and symptoms of other diseases (tuberculosis, malaria, dengue) often complicating the accurate diagnosis for the disease (Sarkar-Tyson & Titball 2009). As such, a study on the burden of melioidosis has estimated approximately 165,000 cases with a mortality rate greater than 50% (89,000 deaths) globally in 2015 (Limmathurotsakul et al. 2016). There is no available data reporting DALYs estimate for melioidosis from WHO GHE 2019 and GBD 2019. Up-to-date presently available global burden of melioidosis in 2015 by Birnie et al. (2019) described an estimated 4.64 million DALYs which surpassed all 16 other NTDs.\n\nLeptospirosis is a zoonotic disease caused by a lethal bacterium of the genus Leptospira (Horwood et al. 2019). The bacteria resides in the hostâs kidney, completing its lifecycle before being shed in the urine. Molecular serotyping studieshave concluded more than 20 Leptospira spp. which can be further segregated into three phylogeny clades of pathogenic, intermediate, and non-pathogenic (Levett 2015). Various wild and domesticated mammals are suitable host reservoirs for Leptospira spp. In the city; rodents are the most important host sources of leptospirosis infection as they can persistently shed pathogenic Leptospira spp. to the environment throughout their lifecycle without any clinical manifestations (Urbanskas, KarvelienÄ & Radzijevskaja 2022). According to Andre-Fontaine, Aviat, and Thorin (2015), Leptospira spp. can survive for up to 20 months at 30°C and up to 10 months at 4°C. Human individuals can contract the illness through direct contact with Leptospira-contaminated urine, water, and wet soil (Sun, Liu & Yan 2020). Pathogenic Leptospira spp. infections may be asymptomatic or exhibit a variety of clinical symptoms, from acute febrile sickness to severely defined multiple organ failures, mimicking symptoms of other threatening diseases such as dengue, influenza, and malaria (Haake & Levett 2015; Sykes et al. 2022). There is no accurate data available regarding the burden of leptospirosis from WHO GHE 2019 and GBD 2019, hence a model study calculated that there are roughly 1.03 million incidents of leptospirosis annually worldwide, of which 5.72% (58,900) result in mortality (Costa et al. 2015). Additionally, those figures were incorporated by Torgerson et al. (2015) to estimate the global burden of leptospirosis in terms of DALYs which were predicted to be at 2.90 million DALYs annually, representing incidence of 41.8 DALYs per 100,000 population (UI 18.1 â 65.5).\n\nMosquito bites from infected females Anopheles spp. deliver the deadly parasites that cause malaria, which is a long-standing disease (Christophers 1951). The causative agent for malaria is a group of unicellular protozoan parasites originating from the Plasmodium genus (Sato 2021). All Plasmodium spp. are capable of infecting malaria but to a specific range of host, with P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi as natural vectors for malaria among humans (Lalremruata et al. 2017). Furthermore, P. falciparum is the most lethal and prevalent malaria parasite in Africa, whereas P. vivax is the most common malaria infection outside of Sub-Saharan Africa (Larson 2019; Liu et al. 2014). In 2020, it was predicted that there were 241 million cases of malaria, resulting in 627,000 deaths (Singh et al. 2022). In the same report, malaria has posed a great threat to about half of the world's population, with sub-Saharan Africa reporting the greatest number of cases and fatalities. The burden of malaria in the WHO African Region is disproportionately high, accounting for 95% and 96%, respectively, of malaria cases and deaths in 2020. In addition, the WHO African Region documented that children under the age of five made up over 80% of all malaria deaths in 2020, making them the most vulnerable group to the disease (Saba, Balwan & Mushtaq 2022). GBD 2019 estimated a significantly higher DALYs burden of 46.4 million years compared to WHO GHE 2019, with age-standardized rates of 667 DALYs per 100,000 (95% UI 337 â 1,150) (Vos et al. 2020).\n\n\nAnalysis of recent literature\n\nIn this section, we explore the applications of ML tools in drug discovery and development, and surveillance and disease management for the aforementioned diseases. Next, we briefly discuss the advances of ML tools in adjacent fields of protein- and antibody-language models, cancer research, and computer vision that can be further leveraged for NTDs research and disease management. Lastly, we discuss steps taken for regional collaboration, data and infrastructure sharing within and around SEA and Western Pacific regions.\n\nThe conventional approach to drug discovery is expensive and takes up a considerable amount of time. Computational approaches to drug discovery using Artificial Intelligence (AI) can resolve both concerns and speed up the process of novel drug discovery. Machine learning (ML) is a subfield of Artificial Intelligence (AI) where sets of data and algorithms (mathematical and statistical) are utilized in search of distinct patterns within the data for a more efficiently accurate downstream analysis (McComb, Bies & Ramanathan 2022). ML in drug discovery and development are carried out by looking for patterns in sets of molecules with drug- and therapeutic-properties to describe in detail their biological activities (Dara et al. 2022). Tasks such as classification (prediction of classes), clustering (grouping of similar data items), and regression (prediction of continuous values) can be performed using ML approaches (Oguike et al. 2022). The role of ML in limiting the spread of deadly diseases (such as disease forecasting, outbreak prediction, disease outbreak detection, and risk prediction) has been detailed reviewed by Alfred and Obit (2021).\n\nWe note that literature mining over the past five years (2017 till 2022) using Google Scholar and Public Library of Science (PLOS) on application of âML tools in drug discovery and developmentâ resulted in articles for dengue, cysticercosis, leptospirosis, and malaria. Literature search did not return relevant articles for four other diseases, namely STHs, rabies, LF, and melioidosis. Nevertheless, literature on âML tools in surveillance and disease managementâ were successfully retrieved for each disease. Moreover, six of the presently listed NTDs (dracunculiasis, lymphatic filariasis, onchocerciasis, schistosomiasis, soil-transmitted helminthiases, and trachoma) can be controlled, eliminated, and prevented through recommended strategic interventions. Efforts from conducting hygiene education programs, innovative and intensified disease management, mass drug administrations (also called preventive chemotherapy), provisioning and educating the principles of safe water, sanitation and hygiene (WASH), vector control, and veterinary public health have helped speed up efforts in eliminating these diseases (Hotez & Lo 2020; Zeynudin et al. 2022). We also suspect the possible fact that since helminthic diseases (LF and STHs in this review paper) can be easily treated with long existing anthelmintic drugs, this did not spark any interest among researchers to develop ML tools in developing a new drug against it (Butala et al. 2021). Moreover, reports on significant decrease of LF and STHs infections were achieved through the combination of strategic interventions as recommended by WHO (Yajima & Ichimori 2021; Zeynudin et al. 2022). In the case of rabies, there is a vaccine available to prevent the infection to both humans and animal companions along with comprehensive surveillance. Lastly, the number of melioidosis cases are comparably lower than any of the diverse group of NTDs despite the high mortality rate of the disease which could be a possible explanation on why no research employing ML tools were invested for the disease. Despite that, the DALYs burden estimates for each of the diseases underlines a pressing need among researchers to develop accurate, sensitive, and specific surveillance, clinical diagnostic, disease detecting, prediction and/or distribution modeling to reach the elimination targets for these diseases.\n\nDengue\n\nDrug Discovery and Development ~ The DENV replication process requires the NS3 protease domain, and the cofactor NS2b which is vital for substrate recognition and complex stability. Both molecules form the NS2b-NS3 protease complex which is a popular target candidate for antiviral drug study due to its key importance for viral replication. However, presently available choices of inhibitors during that time were unsatisfactory due to weak activity or low selective index towards the NS3 active site. Aguilera-Pesantes et al. (2017) utilized ML methods to identify potential residues and sites for drug-like molecule interaction, and bindable sites for drug development through the computational analysis approach for each amino acid in the DENV protease. They used four ML models, Random Forest (RF), Least absolute deviation tree (LAD Tree), voting feature interval (VTI), and multilayer perceptron algorithm (MLP), to classify their predicted data of i.) mutational susceptibility; ii.) residual binding site; iii.) physico-chemical properties; and iv.) computational alanine scanning mutagenesis for protein binding affinity and stability predictions. Each modelâs performance was measured through recall, precision, area under the receiver operating characteristics curve (AUC: area under the ROC) metrics. At the end of their study, MLP-based models yielded the best performance in properly classifying residues interaction with NS3 that would cause major change in activity, moderate change in activity, and residues with similar activity as wild type residues (Aguilera-Pesantes et al. 2017).\n\nThe application of Artificial Neural Network (ANN) to predict Dengue-Human protein interaction type leading to development of antiviral drugs was reported by Jainul Fathima et al. (2019). They trained the model with a dataset made up of 535 non-redundant interactions between 335 different human proteins and ten dengue proteins, each of which is made up of eight attributes and 550 instances, using the Feed Forward Back Propagation Neural Network (FFBPNN) technique. As many as 12 categories of human protein interaction with dengue protein were generated to be selected for attribute selection, and then ranked based on the weights. The prediction accuracy on the test dataset was 98.05%. Two human proteins HBA1 and HSPA5 were discovered to have greater interaction with dengue virus compared to others, plus the NS3 and NS5 dengue proteins were proven to be of therapeutic drug target potential.\n\nKhalid et al. (2020) reported a study that uses ML methods to investigate the biological activities of inhibitor derivatives anti-dengue compounds. They employed an atom-based three-dimensional (3D) QSAR modeling study along with the machine learning software Schrödinger Drug Discovery Suite Phase⢠to investigate the compound's structural features with the anti-dengue activities. As a training dataset, a homologous series of 21 newly discovered 1,3,4-oxadiazole derivatives compounds was used. Using the built-in random selection mechanism of the Schrödinger Phase⢠software, the prepared datasets were then separated into training and test sets, 75% and 25% respectively. Based on the predictability of the biological activities of the test molecules, the model's predictive capacity was found to be adequate with Q2 (R2 Training Set) = 0.73 and Q2 (R2 Test Set) = 0.78. The chemical structural features of the predicted models were used as a benchmark for developing novel 1,3,4-oxadiazole derivatives compounds against dengue virus (Khalid, Rao Avupati & Hussain 2020). Subsequently in the same year, Geoffrey and his colleagues (2022) employed a ML-based AutoQSAR, which encompasses feature selection, QSAR modeling, validation, and prediction to generate drug leads from PubChem database for Dengue and West Nile virurs. The ML-based AutoQSAR algorithm helps to expedite virtual drug screening and identification against Dengue and West Nile virus and also perform automated in silico examination of the drug lead compounds. Readers interested in conducting 3D-QSAR modeling for their research are recommended to opt for Py-CoMFA, an open source web-based alternative (Ragno 2019).\n\nElakkiya Elumalai (2022) published a study in which ML techniques were used to identify and classify peptides as either inhibitory or non-inhibiting of the dengue virus. A dataset of 100 peptides that have been experimentally verified to inhibit the dengue virus and 16 negative datasets from the antiviral peptides database (AVPdb), were both divided into training and testing sets with a 7:3 ratio. Eight different ML algorithm models, including Adaptive Boosting (Adaboost), Bagging, k-Nearest Neighbor (kNN), Logistic Regression (LR), Multi-Layer Perceptron (MLP), Naïve Bayes (NB), Random Forest (RF), and Support Vector Machine (SVM), were used to compare three amino acid descriptors, Amino Acid Composition (AAC), Grouped Amino Acid Composition, Transition, and Distribution (GAAC), and Composition, Transition and Distribution Features (CTD). Five of their best models on training data reported accuracies greater than 85%. The same five models were used for testing in which two models (AAC_RF_model and AAC_k-NN_model) reported accuracy of 85.71%, whereas the remaining models are less than 80% accuracy. Both k-NN and RF algorithms implemented were validated as the best algorithm in achieving the research goal. In addition, their study discovered higher frequency of glycine (G), phenylalanine (F), and tryptophan (W) amino acids found in dengue virus inhibitory peptides (Elumalai 2022).\n\nSurveillance and Disease Management ~ Dengue surveillance is crucial for detecting outbreaks and monitoring disease incidences. Increasing the number of surveillance traps that capture eggs (ovitraps) and ovipositing females (gravid traps) with appropriate larvicide and mosquitocide (Selvarajoo et al. 2022). This is to prevent hatching of eggs or any subsequent production of mosquitoes inside the trap. This method is a two-pronged approach allowing authorities to survey the incidences and population of mosquitoes as well as for vector control. However, counting of both traps requires a group of individuals (insectaries) that possess considerable degree of skill to count eggs and sampling specific stages of mosquitoes (Tsheten et al. 2021). Other than vector surveillance, insecticide resistance in the vector population should be identified via susceptibility bioassays, which should be performed by the governing authorities. Observation of the phenotypic response of mosquitoesâ post-exposure to insecticides should be a sufficient metric in determining presence of insecticide resistance.\n\nControl of dengue is mainly achieved through the cooperation of all walks of life. Common strategies include removal of mosquitoes breeding sources, eliminating container habitats that would collect water that are favorable for oviposition sites and development of mosquito larvae, killing of larval or pupal mosquitoes by applying environmentally friendly insecticides, and usage of spatial repellents (Srisawat et al. 2022). Various strategies of sterile insect technique (SIT) all aimed at causing decline of targeted insect population through the release of sterilized male insects were proven to be a significant measure in controlling mosquito populations (De Castro Poncio et al. 2021; Hugo et al. 2022; Ranathunge et al. 2022). Despite these advances, dengue infection remains largely uncontrollable in both rich and poor populations due to several factors. This was seen in Singaporeâs 15 year-long intensive vector surveillance from the mid-1970s to the late 1980s, where low incidences of dengue were reported. In 1990 onwards, the country faced repeating cyclical epidemics where the largest epidemic occurred back in 2013 with over 22,000 cases despite the continued investment of US$50 million in vector control annually (Molyneux 2019). Hence, there is a need for improving active surveillance aspects and developing drugs for better disease management.\n\nWith respect to the three types of dengue infection that may manifest among patients, Hoyos, Aguilar, and Toro (2022) employed various ML decision support systems to develop an autonomous cycle of data analysis tasks (ACODAT) to help medical personnel in clinical disease management of dengue cases. Large population dataset of approximately 70,000 patients was utilized to train the tool in verifying patient data information, classifying the type of dengue a patient has contracted, and listing the best treatment accordingly. The authors used a MLP with a single layer in the case for ANN and classifier version of SVM. Both ML-based classification models achieved accurate dengue type classification (> 0.97). Then, a genetic algorithm (GA) was utilized to compute information from the classification step to generate the best treatment plan for the patient.\n\nTwo regression-based ML models of multiple linear regression (MLR) and support vector regression (SVR) were employed to predict dengue incidences using information of hospitalized dengue patients, metrological and socioeconomic (Dey et al. 2022). From the study, the SVR models showed higher prediction accuracy of 75% with mean absolute error (MAE) value of 4.95, whereas the MLR model displayed an accuracy of 67% and MAE value of 4.57. Moreover, the models were able to show a positive correlation between the relationship of rainfall index with the incidences of dengue.\n\nPanja et al. (2022) built an ensemble wavelet neural network with exogenous factor (XEWNet) dengue outbreak forecast model based on climatic conditions, capable of performing 75% better when forecasting short-term (26 weeks) and long-term (52 weeks) dengue incidences in subtropical regions that experience moderate to heavy rainfall throughout the year. In their study, a maximal overlapping version of discrete wavelet transformation (MODWT) algorithm in interpreting the time-dependent wavelet and scaling coefficients of rainfall and dengue interrelationship. The authors reported the statistical model employing the auto-regressive neural network (ARNN) in evaluating root mean square error (RMSE) and MAE gave the best forecast accuracies of short-term and long-term dengue outbreaks simultaneously.\n\nSubsequently, Nguyen et al. (2022) developed a dengue fever prediction model employing the DL technique of attention-enhanced long short-term memory (LSTM-ATT) model. In Nguyenâs study, they integrated more environmental variables irrespective of time-lagged (namely evaporation, humidity, rainfall, sunshine hours, and temperature) together with DF incidences for their dengue forecast model compared to Panjaâs and colleagueâs work. The authors proposed LSTM-ATT to be the best performing model when compared to CNN and regular LSTM because of having integrated an additional step of attention mechanism layer right after the LSTM network step (Zhang, Yang & Zhou 2021). When compared against the modern (CNN and LSTM) and traditional prediction models, the LSTM-ATT displayed better prediction performance in terms of low RMSE and MAE values in more than half of their geographical study locations (Nguyen et al. 2022). When selected for outbreak prediction evaluation, the LSTM-ATT model was able to distinguish months of an outbreak from normal at an average accuracy score of 0.99, and the average sensitivity score in detecting dengue outbreak months of 0.70.\n\nSoil-transmitted Helminths (STHs)\n\nSurveillance and Disease Management ~ Since no vectors are involved in the transmission of STHs illnesses among humans, surveillance programs can be separated into (i) prediction- and mathematical-based models covering transmission model, estimating of population at risk, predicting regions in need of MDA interventions, and (ii) active surveillance approach (Chong et al. 2022; Mogaji et al. 2022). Important factors that determine a population's sensitivity to STHs include soil and stool samples (Oyewole & Simon-Oke 2022). The former includes usage of algorithms and large data inputs to make precise decision making of incidences and prevalence for STHs in endemic areas. In contrast, the latter involves the expert skills of laboratory staff and technicians to conduct microscopy-based and molecular-based detection methods to detect presence of helminths and accounts for disease surveillance at the same time. Microscopy-based methods, such as the Kato-Katz and McMaster techniques, are primary diagnostic tests to detect parasites by enumerating the eggs-per-gram (EPG) metric. Although these methods are cheaper, results may vary with an increase of sample size and different survey sites (Afolabi et al. 2022). Hence, molecular-based assays such as PCR, real-time PCR and digital PCR, loop-mediated isothermal amplification (LAMP), and cell-free DNA detection provide a more sensitive, less labor-intensive, and high-throughput detection method despite incurring additional costs for detection and surveillance programmes (Becker et al. 2018; Manuel, Ramanujam & Ajjampur 2021).\n\nMeasures taken in managing cases of STHs are shared among other helminth infections. Implementing MDA programs targeting high-risk groups in endemic tropical and subtropical areas has been recognized to be effective in eliminating STH globally (Alemu et al. 2022). In underdeveloped areas with poor facilities but endemic with STHs, adopting the principles of WASH by providing adequate sanitation, improving waste management facilities, plus public education on hygiene practices and behavioral changes targeted to populations at risk would accelerate the elimination goal (Sato et al. 2019). As for those diagnosed with STHs, proper provision of treatment (stronger combination of antibiotics or surgery to remove the worms) is essential in ensuring a good universal health coverage (Abela-Ridder et al. 2020).\n\nIdentifying disease predictors for frequent incidences of NTDs co-infections (malaria and STHs) were conducted through a gut microbiota study (Easton et al. 2020). RF models were employed to account or classify for T. trichiura egg counts and P. vivax parasitemia. A 10,000 tree RF regression model with 1,345 variables at each split was used to forecast T. trichiura egg counts, while a 10,000 tree RF classification model with 1,346 variables at each split was utilized to assess P. vivax parasitemia. Accordingly, the models reported predictor variables of transforming growth factor β and bacteria taxa when predicting for T. trichiura egg counts or intestinal helminth burden and incidence of P. vivax parasitemia, respectively. The complexities caused by the co-infection are interesting, but the authors noted that longitudinal interventional studies (antimalarial and deworming treatments) are needed to further support or validate the reported results.\n\nDacal et al. (2021) developed a computer vision platform that aids in quantifying T. trichiura infection. From 51 Kato-Katz stool sample slides containing 949 Trichuris spp. eggs, these images were used to train and test the CNN algorithm for automatic assessment. The algorithm showed a mean precision of 98.44% and mean recall of 80.94%. Expanding their model in identifying other helminth eggs, they included positive egg samples for both Trichuris spp. and Ascaris spp. from a co-infection individual and obtained mean precision of 94.66% and mean recall of 93.08%.\n\nSubsequently, Ward et al. (2022) proposed an AI-based digital pathology (AI-DP) device that is tasked for automated scanning and detection of helminth eggs from fecal samples prepared via the Kato-Katz technique. Images from the Kato-Katz technique stool smear slides were collected, annotated for helminths eggs, and then continued with AI training and evaluation. The authors employed the CNN technique as well for their annotated training set. About 90% of the 16,990-image annotated STHs eggs were used to train the DL-based detection model, and then tested with the remaining 10% as test set. Overall, the AI-DP was able to achieve both weighted average precision and weighted average recall of greater than 94%.\n\nSTHs epidemiological risk modeling employing Extreme Gradient Boosting (XGBoost) and Shapley Additive explanation (SHAP) as a means of STHs surveillance (Scavuzzo et al. 2022). A dataset of hookworm infection, environmental variables, and socioeconomic characteristics were supplied to the XGBoost model for analysis in order to model the risk of STHs infection. SHAP was utilized to understand the importance of variables for predictions in the trained model. The final XGBoost model's findings outperformed the conventional statistical models that were compared in their study based on the performance metrics of R2 and Mean Square Error (MSE).\n\nAddressing the issue of complex and time-consuming manual diagnosis of STHs infections, fuzzy c-Mean (FCM) and CNN segmentation technique (ML- and DL-based, respectively) for surveillance of human intestinal parasite ova segmentation were conducted. (Lim et al. 2022). Under the direction of parasitologists, a total of 166 pictures for each species were correctly assembled in order to train both ML-based and DL-based segmentation approaches in identifying intestinal STHs ova. Both segmentation technique models were able to accurately predict helminth species at 97% (FCM) and 100% (CNN). According to a further assessment of the segmentation identification performance (Intersection over Union, IoU) of the two models, the CNN segmentation technique yielded better results than the FCM segmentation technique approach.\n\nRabies\n\nSurveillance and Disease Management ~ Majority of rabies surveillance and monitoring is done by promptly identifying animals exhibiting possible rabies clinical indications, documenting the background of recently deceased companion animals, and keeping track of dog bite incidents (Dadang 2019). In response to the 2017 rabies outbreak that occurred in Malaysia, the authors have listed in detail rabies preventive measures, and control procedures for outbreak (Dadang 2019). Generally, the best method to control cases of rabies is to visit the nearest healthcare or veterinary services to get pre-exposure prophylaxis for both you and your companion animals. Rabies surveillance-diagnosis programs are challenging as the gold standard for rabies lyssavirus detection is direct diagnosis with brain tissue. Next, efforts in controlling the disease are troublesome when incidences of free roaming stray dogs and cats are considered a norm, plus close vicinity to wildlife habitat. Hence, most countries would only initiate a mass dog vaccination program to control the outbreak and to curb the transmission (Molyneux 2019).\n\nSurveillance programs for rabies were proven to be challenging as rabies-infected (rabid) animals can only be identified when clinical symptoms manifest and often lead to death. Hence, drastic actions are needed to reduce rabies mortality. As children are more vulnerable to animals, education is crucial in preventing mortalities from rabies. The death rate among children is significantly decreased by teaching people how to avoid getting bitten and what to do if they fear they have been bitten by a rabid animal. The viral load in the bite wound can be considerably reduced by cleaning it as soon as possible. To break the cycle of rabies transmission, implementing mass vaccination for dogs by health authorities has been recognized to be more cost-effective and protects the well-being of livestock and humans at the same time. As mentioned before, People at high risk of exposure to the rabies virus, such as veterinarians, laboratory workers who work closely with the rabies virus, and those who have been bitten by a potentially rabid animal, are strongly advised to get vaccinated (pre- or post-exposure prophylaxis) (Abela-Ridder et al. 2020).\n\nAs a metric for infectious illness monitoring, information of mononucleotide frequency and dinucleotide biases for viral target hosts prediction study were conducted through alignment methods employing kNN approaches and ML-based predictions of host viruses using SVM approaches (Li & Sun 2018). Here, the conserved nucleoprotein gene sequences of the rabies virus was investigated. In order to pinpoint the host of the virus, the authors implemented a kNN approach that is based on a pairwise distance matrix for both alignment-based and alignment-free distances. For the mono- and dinucleotide frequencies, SVM-based predictions were utilized, and the prediction accuracy was assessed using a leave-one-out cross validation (LOOCV) and N-fold cross-validation. The alignment-based methods gave similar results in being able to accurately predict the virus target host and has outperformed the SVM approach.\n\nSaleh, Medang and Ibrahim (2020) conducted a comparison study on a rabies outbreak prediction model employing deep learning with long short-term memory (LSTM), a type of recurrent neural network, compared to the autoregressive integrated moving average (ARIMA) traditional algorithm model. Predictive capabilities of both models were tested against a dataset of one thousand rabies samples obtained from HealthData.com, and performance metrics were assessed based on RMSE and accuracy. The authors reported lower RMSE value (2.04) and greater accuracy (97.3%) displayed by the LSTM model, compared to the traditional ARIMA model performance of 3.12 RMSE value and only 72.1% accuracy.\n\nIn surveilling the zoonotic potential of novel viruses found in vampiric bats, Bergner et al. (2021) conducted the study while utilizing two ML models that were built by Mollentze, Babayan and Streicker (2021). Novel virus zoonotic potentials were evaluated based on a phylogenetic neighborhood model, followed by a genome composition-based model. These ML models were developed by Mollentze and colleagues employed gradient boosted machine (GBM) classifiers to predict 100 best models out of 1000 iterations (Mollentze, Babayan & Streicker 2021). Bergner and colleagues reported the rabies virus as the only known zoonosis detected from the bats and suggested for molecular surveillance as a measure for the rabies outbreak. As published by Bergner and colleagues, they concluded the genome composition-based ML model worked best (have greater accuracy) in predicting zoonotic potential among novel viruses found in bats (Astroviridae, Coronaviridae, Hepeviridae, Picornaviridae, and Reoviridae) as they were able to gain valuable insights of viral information allowing researchers to prioritize potentially zoonotic novel viruses compared to the phylogenetic neighborhood model.\n\nAs dogs (domestic and stray) are largely responsible for the transmission of rabies virus to humans, Thanapongtharm et al. (2021) employed a ML-based random forest algorithm surveillance study on the spatial population of dogs. The goal of the dog distribution and population RF model was to determine how dog populations and environmental and human population variables interacted. Two RF models were developed by the authors. The first (quantitative RF model) selected for grids with presence of dogs and were modeled as quantitative RF before evaluating the predictive power of the models with correlation coefficient and RMSE metrics. In the second model (binary RF model), grids were defined into presence for dogs or absence for dogs (represented by 1 and 0, respectively), and assessed the predictive power with a correlation coefficient and AUC. The models were able to accurately predict the distribution of owned dogs to stray dogs at a ratio of 6:1 (numerical figures; 12,027:1,868), with approximately 75% of the stray dogs being feral. Moreover, human population factors such as communities, human population density, and proximity to religious praying sites had a high correlation with the number of stray dogs. Association results on the population and spatial distribution of stray dogs can lighten the burden of governing bodies in better managing dog vaccination campaigns to achieve elimination targets of rabies.\n\nCysticercosis\n\nDrug Discovery and Development ~ A multi-epitope chimeric vaccine design study which targets T. solium membrane proteins reported promising results of cellular and humoral immune response stimulation, plus providing protection against both taeniasis and neurocysticercosis (Kaur et al. 2020). Various ML algorithms were utilized in creating the vaccine. The authors used SVM modules to categorize allergic and antigenic proteins based on amino acid and dipeptide composition, the Hidden Markov model to predict B-cell epitope antigenic determinants, and ANN to detect proteasomal C terminal cleavage and T-cell epitopes. Five suitable cell membrane peptides were reported from their vaccine study, capable of stimulating required immune responses against taeniasis and neurocysticercosis.\n\nSurveillance and Disease Management ~ A virtual meeting convened by WHO aimed at reviewing existing diagnostic tools for T. solium before implementing them as public health programs to control the disease (World Health Organization 2022a). In the context of a programmatic surveillance, the WHO recommended inclusion of specific communities or villages that are of a wider geographical area. Purposive sampling should be implemented to target high-risk humans and pigsâ interactions when the community lives in close contact with pigs or pigs roam freely where sanitation is inadequate. All means of diagnostic mapping and monitoring of T. solium presence in humans and pigs did not achieve the required sensitivity due to confirmatory methods via microscopy (humans), meat inspection, and serology testing. Diagnostic tests for public health programs are not well-suited as they are not commercially available and of unsatisfactory sensitivity and/or specificity. Due to the low sensitivity of currently available tests, the WHO urged for an appropriate response even if itâs a weak signal of prevalence detected. Preventive chemotherapy (PC) interventions should be initiated with confirmation of key risk factors of roaming pigs and inadequate sanitation.\n\nAccording to WHO, it is possible to completely eradicate cysticercosis as a public health issue. as interventions in disease management are feasible and achievable. Beginning with T. solium in pigs, improving the well-being quality and management of pig husbandry can effectively break the transmission cycle. Such actions are such as vaccination programs, anthelmintic treatment for pigs, and proper set up of enclosure habitats to prevent access to human feces. To curb incidences of taeniasis and cysticercosis among humans, practice of proper WASH principles along with improved food safety and hygiene standards, as well as sufficient sanitation for the safe disposal of excrement would significantly reduce chances of contracting the diseases. In addition, community health education, MDA interventions, and appropriate case management for taeniasis (medical prescriptions) and NCC (surgery) would aid in removing the disease status as a public health concern (Abela-Ridder et al. 2020).\n\nA study on the cysticercosis diagnosis in pigs using proteomic information from tissues rich in antigens, which was assessed using the leave-one-out cross validation method (Navarrete-Perea et al. 2017). Protein extraction and purification experiments were conducted for the extracted T. solium cysts from the central nervous system and skeletal muscles of infected pigs. Generated proteomic information was then used to train the ML-based model in distinguishing between cysticercosis infected and uninfected pigs. The ML-based model was able to successfully classify cysticercosis infected pigs from non-infected ones, plus displaying similar results to the complex crude cysts extracts technique. When testing their model on human cysticercosis patients, they reported satisfactory performance of their model but noted that the modelâs sensitivity was only at about 75%, and its performance could be improved with a new protein mixture dataset suited for human diagnosis.\n\nGreater number of free-roaming pigs have a positive correlation with the transmission rate of infectious diseases, while not knowing the activity that the pigs had gone through. In tackling such matters, a body harness monitoring device has been developed and tasked in reporting the location and activity of free-roaming pigs which can provide substantial information in understanding where and how the pigs might have gotten the infection plus identifying the place of infection (Haladjian et al. 2017). Here, three ML models (linear discriminant, KNN, and SVM) were employed to classify the activities (walking, eating, and resting) of free-roaming pigs and were then validated through the 10-fold cross validation technique. The authors noted the SVM-model outperformed the other two ML-based models with accuracy, precision, and recall of 95.8%, 75.4%, and 86.6%, respectively.\n\nLymphatic filariasis (LF)\n\nSurveillance and Disease Management ~ Establishment of GPELF by the WHO had set the goal in managing the transmission of LF infections via MDA of anthelmintics and alleviating the sufferings of people affected through morbidity management and disability prevention (MMDP). Surveillance programs for LF were conducted through sentinel and spot-check community surveys. Periodic transmission assessment survey (TAS) measures the impact of MDA interventions and to determine if the level of infection decreases below a target threshold (World Health Organization 2022b).\n\nMDA intervention to halt the transmission of infection through WHO-recommended prescriptions of albendazole, diethylcarbamazine, and ivermectin are strategies for managing LF disease. Usage of insecticide-treated bednets is recommended as a means of vector control in household environments. Application of WASH could be used to guarantee correct sanitation procedures to limit vector breeding sites and hygiene treatment of afflicted limbs for morbidity control. As LF is known to cause significant physical impairment, health authorities must ensure essential care is given to patients. Examples include skin care, exercise, and elevation to stop the progression and severity of lymphedema, treatment for adenolymphangitis flare-ups, hydrocele surgery, and encouraging community cooperation to finish the course of treatment and deal with its physical and psychological repercussions (Abela-Ridder et al. 2020).\n\nAnalysis of epidemiological and socio-economic data to predict LF were conducted by employing ML techniques such as classification and regression tree (RT), gradient boosting machine (GBM), J48 algorithm, JRip algorithm, logistic model tree (LMT), probabilistic neural network (PNN), and NB (Kondeti et al. 2019). In order to eliminate biases that are present in the dataset, the authors combined socioeconomic and epidemiological data before using feature selection and gain ratio feature selection to pick out pertinent features for the prediction model. The data is then partitioned (training, testing, and validation) and experimented under the 10-Fold Cross Validation framework while applying oversampling and undersampling methods to balance the dataset. The performance of the ML-based prediction models was then assessed by sensitivity, specificity, AUC, and accuracy criteria. According to the authors, the J48-based prediction model produced an AUC value of 62% and 23 additional classification rules based on six features, whereas the NB-based prediction model produced the best sensitivity and AUC (64%) results when using gain ratio feature selection and at 400% oversampling. The development of early warning systems to better apply prevention and control measures in managing LF disease within the community are a few of the benefits from both these ML-based prediction models.\n\nElvana and Suryanto (2022) trained a CNN-based model with the Image Processing and Data Augmentation approach to identify parasitic worms on a dataset of 210 microfilarial images. With models like VGG-16, ResNet-50, and Inception-v3 that had previously been trained with a simple 8 Convolutional layer CNN model, the authors performed transfer learning for the CNN model. The CNN was able to recognise LF worms from digital photographs with a 70% accuracy rate, even in the presence of noisy images of blood cell images during the training process.\n\nA recent study by Dickson et al. (2022) investigated the potential of diagnostic testing scenarios surveillance after MDA campaigns against LF in detecting transmission and prevalence of the disease using a Bayesian network framework. The effectiveness of several infection markers in detecting signs of transmission was assessed by using a Bayesian network framework using antigen- and antibody-based data (Wb123 Ab and Bm14 Ab). The algorithm's ability to compare the probability of a missed positive LF result with various diagnostic testing situations and evaluate the impact of numerous participant characteristics led the authors to choose a Bayesian network analysis in this case. The network performances were evaluated in a criteria of sensitivity, specificity, True Skill Statistics, and AUC. According to the Bayesian network model, a sizable fraction of LF-positive cases went undetected by antigen- and antibody-based tests on their own. The most sensitive indication of present or previous LF infection diagnosis came from antigen-antibody combination testing (antigen plus Bm14 Ab). Hence, to increase the sensitivity of transmission surveys and prevent sudden and premature termination of MDA campaigns against LF, the combination of antigen plus Bm14 Ab were proposed for inclusion in post-MDA surveillance.\n\nMelioidosis\n\nSurveillance and Disease Management ~ A ML-based Raman spectroscopic assay was developed to identify B. pseudomallei and Burkholderia mallei strains (Moawad et al. 2019). To train the algorithm, 12 B. mallei strains, 13 B. pseudomallei strains, and 11 other Burkholderia spp. strains were prepared. Physical recording of the Burkholderia spp. Raman spectra were analyzed by the SVM algorithm together with information of the principal component during the Raman spectroscopic assay preprocessing step. The SVM Raman spectroscopic assay was also trained to produce three Burkholderia spp. classification models of (i) pseudomallei-mallei-thailandensis complex from cepacian-glathei-phytofirmans complex, (ii) identifying species of B. mallei, B. pseudomallei, and B. thailandensis accurately from one another, and (iii) identifying species of joined B. cepacian complex and B. glathei from B. phytofirmans. In each of the models, the SVM had identified the assigned bacterial complex and species accurately (>90%), except for the identification of cepacian-glathei-phytofirmans complex group, and B. thailandensis (65%). When validating the performance and sensitivities of the SVM-based Raman spectroscopic assay with unknown Burkholderia strains, sensitivities greater than 80% were obtained.\n\nXu et al. (2021) developed a SVM-based model tasked in detecting clinical septicemic melioidosis infection. Obtaining their data from the human peripheral blood microarray dataset, as many as 69 patients with septicemic melioidosis and a mix total of 175 non-septicemic melioidosis (healthy, type2 diabetes, recovered from melioidosis, and septicemic from other organism) were used to train the SVM-based detection model. When testing against the instance of detecting B. pseudomallei from a mixed group of healthy, type 2 diabetes, and recovery dataset, the SVM classifier yielded sensitivity and specificity of 0.988 and 1.000 respectively. When testing against the instance of detecting B. pseudomallei from other infection dataset, the SVM classifier yielded sensitivity ranging 0.857 to 1.000, and specificity of 0.889 to 1.000. A last validation of B. pseudomallei detection from combination of all health data plus modified infection dataset generated mean sensitivity and specificity of 0.962 and 0.979 respectively.\n\nLeptospirosis\n\nDrug Discovery and Development ~ Abdullah et al. (2021) studied the identification of a suitable Leptospira spp. multiepitope-based vaccine candidate which utilized two ML programs, namely Vaxign-ML and C-ImmSim. In his study, all protein antigens have a protegenicity score greater than 90% signifying as effective antigens for vaccine developments, and simulations from C-ImmSim showed diverse immune reactions of the vaccine construct indicating promising subunits of multiepitope vaccine candidate for immunity against Leptospira spp. Infections.\n\nVaxign-ML is a reverse vaccinology (RV) tool that uses supervised ML to predict the rank score (also known as protegencity) of bacterial protective antigens (BPAgs) using a training set of viral and bacterial antigens (Ong et al. 2020). Out of five additional ML techniques used to develop Vaxign-ML, extreme gradient boosting (XGBoost) was found to be the most effective when using nested 5-fold cross-validation (N5CV) and leave-one-pathogen-out validation (LOPOV) evaluation methods. Set as the benchmark against five other existing programs and methods, Vaxign-EGB-ML displayed satisfactory results outperforming four programs. Final validation on external data sets of clinical trials or licensed vaccines reported ranked calculation of best top 10% BPAg candidates for 20 proteins. Next, the immune simulation study server C-ImmSim uses position-specific scoring matrices and machine learning techniques to seek peptides with epitopes and other immunological interactions (Rapin et al. 2010). The program combines a mesoscopic scale simulator of the immune system with a set of agent-based class computational models to predict molecular-levels of major histocompatibility complex-peptide binding interactions and neural networks for prediction of epitopes.\n\nSurveillance and Disease Management ~ To investigate the spatial distribution of human leptospirosis, Mohammadinia et al. (2019) have employed the ANN, geographically weighted regression (GWR), generalized linear model (GLM), and SVM approaches to model and predict the disease based on environmental parameters of temperature, precipitation, humidity, elevation, and vegetation. All four models were assessed based on mean square error (MSE), mean absolute error (MAE), mean relative error (MRE) and R2. The authors reported that the GWR-based model displayed the best performance in the prediction of leptospirosis, followed by SVM, GLM, and ANN. It was also discovered that temperature and humidity parameters had a great influence on the distribution of leptospirosis among humans.\n\nPredictive risk maps of leptospirosis distribution employing SVM and MLPNN algorithms were conducted by Ahangarcani et al. (2019). As they evaluated the model's performance using the Kappa coefficient and AUC metrics, they looked at the association between altitude, average humidity, average temperature, days below 0°C, land cover, rainfall, slope, and leptospirosis incidents from the previous year. Incidences of leptospirosis in prior years were positively correlated with rainfall, average humidity, and average temperature, but negatively correlated with altitude, slope, land cover, and days below 0°C. Both SVM- and MLPNN-based predictive models displayed satisfactory results with Kappa coefficient and AUC greater than 83% and 0.84, respectively.\n\nA subsequent study in identifying the relationship between the occurrence of leptospirosis with exploratory data analysis of temperature, rainfall, and relative humidity (Rahmat et al. 2020). The authors used an ANN method using back-propagation training, optimization of hidden layers, and hidden nodes to categorize a combination of selected features into determining the presence or absence of diseases. Performance measurement of the ANN-based leptospirosis prediction is evaluated by the modelâs accuracy, sensitivity, and specificity. The ANN model produced the maximum accuracy, sensitivity, and specificity of 84.0%, 86.4%, and 79.3% when measuring the robustness of the model (AUC metric) using a randomized dataset. Additionally, it was reported that using exploratory data methodologies improved the leptospirosis predictive model's accuracy from 13.3% to 31.3%. The weekly average temperature and weekly rainfall total amount at lags of 16 weeks and 12 to 20 weeks, respectively, were found to have a significant link with the incidences of leptospirosis.\n\nMalaria\n\nDrug Discovery and Development ~ A recent study by Mswahili et al. (2021) developed and compared the performance of five ML models to predict antimalarial bioactivities against P. falciparum. They trained ML models of artificial neural network (ANN), SVM, RF, extreme gradient boost (XGB), and LR over a data set of 4,794 antimalarial drug candidate compounds (2,070 active and 2724 inactive molecules). The Recursive Feature Elimination (RFE) wrapper-based algorithm that treats feature selection as a search problem and the K-best filter-based algorithm that selects potential features according to a particular function were chosen as feature selection algorithms for performance examination and comparison. K-best was adopted as an accuracy metric whereas RFE was viewed as an efficiency metric. Based on the two metrics, they found that XGB, ANN, and RF models gave the best three accuracies in finding new antimalarial drug formation without losing too much precision.\n\nApichat Suratanee and colleagues reported the use of four ML classification algorithms, namely NB, NN, RF, and SVM, to investigate protein-protein interaction (PPI) networks for human and malarial parasites retrieved from STRING database (version 11.0), in order to identify new human proteins associated with malaria as a means of developing additional drugs against the disease (Suratanee, Buaboocha & Plaimas 2021). A total of 12,038 human proteins with 313,359 interactions and 1,787 P. vivax proteins with 11,477 interactions were used to train the ML models. While examining the five topological features, including (i) betweenness centrality, (ii) closeness centrality, (iii) degree, (iv) eccentricity, and (v) Kelinberg's hub centrality, they built a heterogeneous network connecting human-human protein interactions and P. vivax-P. vivax protein interactions with the human-P. vivax protein associations. Next, they applied ten 10-fold cross-validations to each algorithm to produce performance metrics of a ROC curve with an AUC, with the RF method coming out on top (AUC of 0.85) and being followed by the NN, SVM, and NB algorithms (AUCs of 0.79, 0.77, and 0.74 respectively). By calculating the top-ranking score for each human protein using the RF classifier's greatest performance and results, the authors were able to acquire 411 human proteins. Subsequent functional annotation of the proteins revealed previously reports of promising candidates for multistage targets for malaria therapy.\n\nUtilizing solely the peptide sequence data, an interpretable scoring card system was employed to pinpoint the antimalarial activity (Charoenkwan et al. 2022). The authors trained an iAMAP with SCM-based predictor with eight other conventional supervised classifiers of decision tree (DT), k-nearest neighbor (KNN), logistic regression (LR), multilayer perceptron (MLP), naïve Bayes (NB), partial least squares regression (PLSLR), random forest (RF), and support vector machine (SVM) with nine conventional feature descriptors namely amino acid index (AAindex), PCP, amino acid composition (AAC), composition, transition and distribution (CTD), CTD-composition (CTDC), CTD-distribution (CTDD), CTD-trancomposition (CTDT), dipeptide composition (DPC), and tripeptide composition (TPC). The training data set consists of 139 positive and 2,135 negative molecules and the test set of 139 positive and 677 negative molecules. The 10-fold cross-validation approach was used to refine the program after initially estimating the propensities of 20 amino acids and 400 dipeptides. Then, estimated propensities were used to choose significant physicochemical features, and the 400 dipeptides' best propensities were used to construct the final prediction model (iAMAP-SCM). Scoring based on maximum accuracy (ACC) and Matthewâs coefficient correlation (MCC), iAMAP-SCM was reported to achieve scores of 0.957 and 0.834 respectively and outperformed the other three classifiers employed, when the model was screened independent test datasets for validation. The application of AI and ML methods to drug discovery and development of malaria have also been reviewed in several recent papers (Oguike et al. 2022; Winkler 2021).\n\nSurveillance and Disease Management ~ Classification of clinical malaria using ML approaches based on hematological parameters is beneficial in improving disease management (Morangâa et al. 2020). The authors employed six approaches to accurately categorize malaria outcomes into uncomplicated malaria, non-malarial infections, and severe malaria. ML algorithms in this study includes ANN, DT, multiple adaptive regression splines (MARS), partial least squares logistic regression (PLSLR), RF, and SVM were all fine-tuned into their best performance state according to the algorithm's kernel. ANN algorithm was further developed into three different models used for multi-classification (among all three malaria categories) and two binary classification (between two malaria categories) of the three categories, and then proceeded with RF algorithm for confirmation of clinical malaria category classification. To measure the performances of each model, metrics such as the accuracy, AUC, confusion matrix, F1 score, and precision and recall were used. All the ML-based models were able to accurately (0.78 to 0.86) classify clinical malaria from non-malarial infections, with SVM and ANN generating the best overall classification outcome. According to an assessment of ANN classification models for clinical malaria, it can distinguish between simple malaria, non-malarial illnesses, and severe malaria with an accuracy better than 0.8 and diagnostic capacities (measured by AUC) greater than 0.86. The three models created by ANN were subjected to RF analysis, which revealed that all three models had accuracy levels higher than 0.76. Platelet counts and red blood cell counts were found to be the most crucial features for categorizing the clinical malaria categories.\n\nOkagbue et al. (2021) employed six ML algorithms, namely Adaboost, DT, kNN, LR, NN, and RF, to build malaria diagnosis models using a sample size of 337 composed of age and sex data, and 15 disease symptoms. Performance of all six models in correctly classifying positive-for-malaria from negative-for-malaria, are as follows; 68.2% (LR); 71.8% (kNN); 89.6% (DT); 92.6% (RF); 95.8% (NN); and 100% (Adaboost). Investigating the effects of including age and sex data on the performance of all six classification models, a second run solely utilizing 15 symptoms was conducted by the authors. A slight decrease in precision and accuracy performance were noted across all models. Performance for all six models remains unchanged with Adaboost being the best performing model and LR performing the least, as ranked previously in ascending order. Here, the Adaboost-based model was still the best performing model with classification accuracy of 98.2%, precision of 96.6%, and error rate of 1.8%. The authors noted that the Adaboost-based model agrees prediction accuracy from similar studies (Alzheimerâs diagnosis from MRI scan, breast cancer diagnosis, diabetes diagnosis, and prostate cancer diagnosis), and results outcome with the inclusion of sex and age data generate better AUC metrics with zero error-rate (misclassification of positive- or negative-for malaria).\n\nA recent study employing ML- based models to predict risk for malaria based on mutation location were published by Tai and Dhaliwal (2022). In this research, researchers examined genetic variation data from 20,817 people from the Malaria Genomic Epidemiology Network (MalariaGEN). Based on 104 feature sets of malaria genetic markers, three ML-based models of LightGBM, Ridge Regression, and SVR were built to predict malaria risk. LightGBM was the top-performing ML-based model (MAE score of 6.39E-01 on all 104 features), and it also outperformed the other two ML-based models when it came to performance results when predicting with fewer features information. Additionally, 50% fewer features (52 features are enough to replace the 104 features used in the previous malaria risk prediction) were reported to be sufficient in predicting malaria risks.\n\nIn summary, nationally representative survey programs suited for the geographical and environmental etiological factors for each respective country. For example, DHS or demographic and health surveys, could provide a suitable platform for ongoing disease surveillance programs. Picking the proverb âprevention is better than cureâ, core strategic interventions together with disease management will better facilitate in eliminating the prevalence and transmission of diseases, and at the same time decrease the morbidity and mortality inflicted.\n\nThe majority of studies employing ML models to discover novel drug candidates for NTDs (but not all), have been published in the past two decades. Similarly, applications of ML in cancer research have been in practice since the early 2000s (Bertsimas & Wiberg 2020). Research domains where ML-based methods can be employed in cancer biology includes genomics, proteomics, metabolomics, epigenetics, transcriptomics, and system biology (Kourou et al. 2021; You et al. 2022). In the computer science bibliography of the Digital Bibliography and Library Project (DBLP) and the biomedical repository PubMed, literature mining on ML-based studies on cancer diagnosis, patients' classification, and prognosis (excluding reviews and technical reports) between 2016 and 2020 were able to retrieve 921 and 165 studies, respectively.\n\nDespite the advances in chemotherapy and immunotherapy, early detection of cancer increases oneâs survival rate tremendously. Technological innovations have sprouted a new branch of AI known as computer vision (CV), which will significantly lighten the burden of physicians and radiologists when it comes to interpreting an MRI or histology slide for the presence of a tumor. Successful early diagnosis of breast cancer using convolutional neural network (CNN) to analyze histopathological images were reported, with additional validation from other researchers of promising and accurate diagnostic capabilities in analyzing imaging slides with the use of deep CNN architectures. Compared to detection at later stages, the cancer would have metastasized, spread to vital organs where surgery may not be feasible. As reviewed by Kourou et al. (2021), most cancer ML-based studies on cancer detection and diagnosis centered around developing DL architectures of automated diagnostic models aiding radiologists and physicians to handle and better identify or characterize imaging data (input) from computed tomography (CT), magnetic resonance imaging (MRI), X-ray radiography, and positron-emission tomography (PET). Another short example is the development of an image-based lung cancer detection model, whereby a region-based CNN model trained with 42,290 whole-CT lung scans has outperformed the average radiologists at malignancy risk-prediction and achieved AUC score greater than 95% when validated with 1,139 clinical cases (Ardila et al. 2019). An overview of the application of AI in identifying cancer targets and drug discovery has been reviewed (Alqahtani 2022; Shao et al. 2022; Taylor 2020; You et al. 2022).\n\nAdvances in the field of AI, typically ML and DL methods, were utilized to develop language models to predict proteins. Algorithms from these methods were employed to process the efficiency and quality of the natural language processing (NLP). To develop a protein language model (PLM), large text (protein sequences from large databases) is given as input to train the prediction of masked or missing amino acids (Bepler & Berger 2021; Ofer, Brandes & Linial 2021; Rives et al. 2021). Literature findings on advances of protein embeddings displayed great performances in predicting secondary structure and subcellular location comparable to other methods that employ evolutionary information from MSA inputs, substituting sequence similarity for homology-based annotation transfer, and predicting mutational effects on protein-protein interactions (PPI) (Alley et al. 2019; Heinzinger et al. 2019; Littmann et al. 2021; StÀrk et al. 2021; Zhou et al. 2020).\n\nVariant Effect Score Prediction without Alignments (VESPA) is able to predict sequence residue conservation and single amino acid variants (SAV) almost as comparatively accurately to other existing methods (DeepSequence, ESM-1v, and GEMME) without employing multiple sequence alignment (MSA) approach to learn more about the functional, structural, sequence conservation and evolutionary information that the organism or gene underwent (Marquet et al. 2022). PLM-based structure prediction models, such as AlphaFold2 (AF2) and RoseTTAFold, successfully solved the atomic-resolution structure prediction problem by using MSAs and templates of related protein structures to provide the highest possible structural prediction performance (Baek et al. 2021; Jumper et al. 2021). Evaluating the performance of MSA-based LMs in isolating coevolutionary signals encoding functional and structural constraints from phylogenetic correlations, Lupo, Sgarbossa, and Bitbol (2022) discovered less phylogenetic deterioration from MSA contact inference plus greater structural contacts accuracy compared to the Potts model despite the MSA Transformer being pre-trained with a minimized diversity dataset. Advancing the MSA approach, an end-to-end differentiable recurrent geometric network (RGN2) for structure prediction of single protein sequences outperforming MSA-based tools (AF2 and RoseTTAFold) was published by Chowdhury et al. (2021) which employed: (i) AminoBERT PLM, which learns latent structural information from millions of misaligned proteins; and (ii) geometric modules representing the Cα backbone geometry. Subsequently, Lin et al. (2022) developed a similar program named ESMFold capable of competing with AF2 and RoseTTAFold in atomic level protein structure prediction accuracy solely with information on individual sequences of rare proteins. Absence of MSA-based elements have significantly shortened the process of protein structure prediction as much as up to six-fold faster than existing tools.\n\nAntibodies represent a unique group of proteins, development of an antibody language model (ALM) for prediction would definitely outperform a trained protein language model that covers a holistic range of protein. ALM AbLang outperformed both IMGT germlines and protein language model ESM-1b in terms of a faster completion time and capability in restoring missing residues of antibody sequences retrieved from the Observed Antibody Space (OAS) database (Olsen, Moal & Deane 2022). Ablang was able to scrutinize better by separating the sequences based on V-genes into smaller clusters, segregate and classify accurately between naïve and memory B-cells, and restored missing residues of sequences with 15 missing residues at the N-terminal without any additional germlines information. Another antibody LM, Antibody-specific Bidirectional Encoder Representation from Transformers (AntiBERTa), was reported to outperform two existing PLMs (ProtBert and Sapiens) and exhibited better B cell receptors representation when compared to ProtBERT that was assigned with a smaller dataset (Choi 2022; Leem et al. 2022). The authors described self-attention changes as the element of AntiBERTa allowed it to correctly predict paratope positions of both CDR and non-CDR positions, better distinguish naïve and memory B-cells than two other PLMs and focused on what is functionally important for specific binding. Interested readers are referred to the design methods of a linguistic-based formalization of the antibody language (Vu et al. 2022). Readers interested in methods of antibody language models are referred to a comprehensive review that discuss the progress, methods, and challenges (Akbar et al. 2021, 2022).\n\nIn summary, the applications of ML tools in both neglected diseases and cancer, computer vision, and protein language research are very similar with one another. Both fields utilized ML tools for drug discovery and development, novel drug target sites, disease surveillance, prediction, detection, and disease management. The main factor that gaps both fields from one another is that researchers are more invested in advancing breakthroughs in cancer, computer vision, and protein language. Moreover, this research have been primarily driven by the accumulation of large training datasets and the development of highly sophisticated deep learning architectures. Typically, large attention-based models are trained on datasets in the order of 106â107 data points. In contrast to NTD research where datasets remain restricted at the scale of 102â103 (up to five orders of magnitude lower), shallow ML-methods, namely LR, RF, SVM, among others, are more prevalent. The lack of large datasets restricts the widespread application of large deep learning models for the discovery of new NTD therapeutics and thus hampers the potential for efficient management and eradication of these diseases.\n\nThe Southeast Asian countries are strategically located and exceptionally diverse in culture. Apart from the geographic proximity of Member States of the ASEAN regional association, the countries share a few other similarities of having densely populated communities, mineral-rich economies that open throughout the globe, and share similar tropical and subtropical climates. Hence, when thereâs a disease outbreak reported among any of the SEA countries, chances of the imported disease to neighboring countries are very high. Hence, there is a need to circumvent the matter through regional collaboration, and data plus infrastructure sharing among the SEA countries.\n\nAs previously described, the SEA region is endemic to vector-borne diseases such as arboviral diseases (dengue, LF, and malaria), leptospirosis, cysticercosis, and rabies. These diseases require up-to-date, robust, and comprehensive information on presence, species-strain diversity, ecology, environmental and geographical information regarding the organisms that carry and transmit the infectious agents. As such, a few platforms had been established, all aimed at tackling these vector-borne diseases at a regional scale, may it be as an open-access publicly available database, or an online platform aimed at collecting data, analyzing them, and providing technical skills and collaboration with both local and global stakeholders. Two very notable and frequently visited database for health metrics and disease related data retrievals that have been actively mentioned throughout this review are none other than WHOâs Global Health Observatory (GHO) and Global Health Data Exchange (GHDx) data catalog that is created and supported by the Institute for Health Metrics and Evaluationâs (IHME) at the University of Washington, where both the Global Health Estimate (GHE) 2019 data and Global Burden of Disease Study (GBD) 2019 could be retrieved respectively by interested readers. Next, the European Centre for Disease Prevention and Control (ECDC) is an open-access database on dengue surveillance, threats, and outbreaks governed by the European Union. The database consists of almost all NTDs and other diseases that are of public health concern. Like MAP, ECDC serves as a platform that collects, analyses, shares, and provides infectious disease data and guidance as a means of assessing disease risks, preventing, and responding to outbreaks and other public health threats. Another publicly available but somewhat geographically irrelevant to SEA region is the WHO-driven Expanded Special Project for the Elimination of NTDs (ESPEN). However, the ESPEN portal only contains survey data sets of NTDs in Africa in response to 39% of the global NTDs burden that occurs in Africa. Undeviating from the WHO goal of accelerating the elimination and eradication of NTDs, ESPEN serves as a portal to aid governing bodies and health officials to rapidly strategies and deploy NTDs interventions to reach key targeted communities.\n\nThe Global Alliance for Rabies Control (GARC) is the leading international non-profit organization dedicated to eradicating canine rabies. GARC collaborates with global stakeholders, governments, and local partners to increase public awareness of rabies, promote teamwork, and develop the data required to increase political commitment and funding. Their main team of nine members work across three established work networks of ARACON (Asian Rabies Control Network), MERACON (Middle East, Eastern Europe, Central Asia and North Africa Rabies Control Network), and PARACON (Pan-African Rabies Control Network) as an effort to end rabies. International body responsible for infrastructure sharing in combating LF is the Global Alliance to Eliminate Lymphatic Filariasis (GAELF). Having great breakthroughs by GPELF in eradicating LF as mentioned previously, GAELF is a steering body aimed at bringing relevant partners to support the GPELF established by WHO via political, financial and technical resources mobilization. The Global Atlas of Helminth Infections (GAHI) is an open-access database that details the global and geographical distribution of three neglected tropical worm-borne diseases: LF, STHs, and schistosomiasis. The platform was developed by London Applied & Spatial Epidemiology Research Group (LASER) at the University of London. All GAHI resources are available on an open access basis but up till the year 2015 only.\n\nIn response to the neglect of melioidosis, there is an active website Melioidosis.info which serves as an online-platform for reporting melioidosis cases and for disseminating information of melioidosis for the public, researchers and health policy makers. Researchers or health authorities with culture-confirmed melioidosis cases and deaths, backed with institutional support can contribute to the platform by uploading melioidosis cases and serological information. The teamâs aim is to allow local and global policy makers to easily pinpoint incidences of melioidosis cases geographically and to identify institutions that are capable of diagnosing the disease. In response to the outbreak of leptospirosis, Global Leptospirosis Environment Action Network (GLEAN) was established to lessen the impact of leptospirosis on the planet by improving our knowledge of the connections between the disease's occurrence and various associated factors, such as biological, demographic, environmental, ecological, and economic factors, delivering more prompt warnings of outbreaks, and identifying prevention and control strategies. In order to predict geographical limitations, prevalence, and endemicity of malaria in every region of the world, the Malaria Atlas Project (MAP) is an open-access database and the WHO collaborative platform for geospatial disease modeling. Solely by handling data, collaboration, analytics, and engagement elements by members of MAP, they have successfully helped (i) to generate malaria mapping risk (infection prevalence, incidence rates, and mortality estimations) at national and global levels; (ii) forecast annual global malaria burden; (iii) tracked interventions coverage (malaria drugs, diagnostics, and vector control); (iv) employed statistical models to measure the effectiveness of currently available control strategies against malaria; (v) developing tools to support efficient commodities planning to ensure enough resources were prepared to protect a population; and (iv) strengthening skills among researchers and technicians in malaria analytics.\n\nData and infrastructure sharing is undeniably crucial for NTDs since the scale of publicly available datasets for NTDs research is restricted. Efforts displayed by each governing body in maintaining and keeping up-to-date open-access databases or infrastructure and technical outreach organizations are to ensure that every country would have the latest disease intelligence and technical skills in order for effective surveillance, preventive, and disease management control to be executed according to each countryâs governing leadership. Importance of having centralized data sharing at a regional scale has been highlighted in a study by Alemu et al. (2022). With access to publicly available standardized survey and treatment coverage data, which was at first unavailable probably due to absence of reports by the countryâs Ministry of Health to the WHO, they were now able to access ample amounts of collected evidence pointing to the advantages of school-based deworming programs and LF MDA campaigns.\n\n\nConclusion\n\nNTDs impact nearly 2 billion people especially in countries with developing economies such as countries in Southeast Asia region causing reduction in productivity and substantial accumulation of Disability-Adjusted Life Years, DALY. Machine learning has been widely applied in the fields of NTDs for drug discovery and development, plus surveillance and disease management. However, the application of machine learning in NTDs research is hampered by the limited amount of data, the absence of centralized/standardized collaborative framework and the general lack of attention from public and private stakeholders alike when compared to other fields. To unleash the full potential of machine learning to elevate the state-of-the-art NTDs surveillance, management, and treatment, increased investment in terms of research funding, public-private collaborative initiative, data accumulation and sharing are desperately needed.",
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Critical Care Clinics. 2013; 29(2): 259â277. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh J, Arora MS, Sharma S, et al.: Modeling the variable transmission rate and various discharges on the spread of Malaria. Electronic Research Archive. 2022; 31(1): 319â341. Publisher Full Text\n\nSrisawat N, Thisyakorn U, Ismail Z, et al.: World Dengue Day: A call for action. PLoS Neglected Tropical Diseases. 2022; 16(8): 2â10. Publisher Full Text\n\nStÀrk H, Dallago C, Heinzinger M, et al.: Light attention predicts protein location from the language of life. Bioinformatics Advances. 2021; 1(1). PubMed Abstract | Publisher Full Text | Free Full Text\n\nSun AH, Liu XX, Yan J: Leptospirosis is an invasive infectious and systemic inflammatory disease. Biomedical Journal. 2020; 43(1): 24â31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuratanee A, Buaboocha T, Plaimas K: Prediction of Human-Plasmodium vivax Protein Associations From Heterogeneous Network Structures Based on Machine-Learning Approach. Bioinformatics and Biology Insights. 2021; 15: 117793222110133. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSykes JE, Reagan KL, Nally JE, et al.: Role of Diagnostics in Epidemiology, Management, Surveillance, and Control of Leptospirosis. Pathogens. 2022; 11(4): 1â24. Publisher Full Text\n\nTai KY, Dhaliwal J: Machine learning model for malaria risk prediction based on mutation location of large-scale genetic variation data. Journal of Big Data. 2022; 9(1). Publisher Full Text\n\nTaylor B: Artificial Intelligence in Oncology Drug Discovery and Development. Artificial Intelligence in Oncology Drug Discovery and Development. 2020.\n\nThanapongtharm W, Kasemsuwan S, Wongphruksasoong V, et al.: Spatial Distribution and Population Estimation of Dogs in Thailand: Implications for Rabies Prevention and Control. Frontiers in Veterinary Science. 2021; 8(December): 1â12. Publisher Full Text\n\nTorgerson PR, Hagan JE, Costa F, et al.: Global Burden of Leptospirosis: Estimated in Terms of Disability Adjusted Life Years. PLoS Neglected Tropical Diseases. 2015; 9(10): e0004122. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsheten T, Gray DJ, Clements ACA, et al.: Epidemiology and challenges of dengue surveillance in the WHO South-East Asia Region. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2021; 115(6): 583â599. Publisher Full Text\n\nUrbanskas E, KarvelienÄ B, Radzijevskaja J: Leptospirosis: classification, epidemiology, and methods of detection. A review. Biologija. 2022; 68(2): 129â136. Publisher Full Text\n\nVinkeles Melchers NVS, Stolk WA, van Loon W , et al.: The burden of skin disease and eye disease due to onchocerciasis in countries formerly under the african programme for onchocerciasis control mandate for 1990, 2020, and 2030. PLoS Neglected Tropical Diseases. 2021; 15(7): 1â18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVos T, Lim SS, Abbafati C, et al.: Global burden of 369 diseases and injuries in 204 countries and territories, 1990â2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet. 2020; 396(10258): 1204â1222. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVu MH, Akbar R, Robert PA, et al.: Advancing protein language models with linguistics: a roadmap for improved interpretability. arXiv. 2022; 1â26.\n\nWard P, Dahlberg P, Lagatie O, et al.: Affordable artificial intelligence-based digital pathology for neglected tropical diseases: A proof-of-concept for the detection of soil-transmitted helminths and Schistosoma mansoni eggs in Kato-Katz stool thick smears. PLoS Neglected Tropical Diseases. 2022; 16(6): 1â16. Publisher Full Text\n\nWHO: Taeniasis/cysticercosis. Fact sheets. 2014; 4.\n\nWibawa T, Satoto TBT: Magnitude of Neglected Tropical Diseases in Indonesia at Postmillennium Development Goals Era. Journal of Tropical Medicine. 2016; 2016.\n\nWinkler AS, Klohe K, Schmidt V, et al.: Neglected tropical diseases â the present and the future. Tidsskrift for Den Norske Legeforening (born 1971). 2018; 138. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: First WHO report on neglected tropical diseases: working to overcome the global impact of neglected tropical diseases. World Health Organization; 2010; 1â184.\n\nWorld Health Organization: Tenth report of the Strategic and Technical Advisory Group for Neglected Tropical Diseases (STAG-NTDs).2017.\n\nWorld Health Organization: Global Health Estimates 2020: Disease burden by Cause, Age, Sex, by Country and by Region.2020; 2000â2019.\n\nWorld Health Organization: Virtual Meeting of Regional Technical Advisory Group for dengue and other arbovirus diseases (October).2021; pp. 4â6.\n\nWorld Health Organization: Taenia solium - Use of existing diagnostic tools in public health programmes. World Health Organization; 2022a.\n\nWorld Health Organization: Global programme to eliminate lymphatic filariasis: progress report, 2021. WHO Weekly Epidemiological Record; 2022b.\n\nWorld Organisation for Animal Health: Rabies Technical Disease Information.2008; pp. 1â4.\n\nXu K, Lian F, Quan Y, et al.: Septicemic Melioidosis Detection Using Support Vector Machine with Five Immune Cell Types. Disease Markers. 2021; 2021: 1â9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYajima A, Ichimori K: Progress in the elimination of lymphatic filariasis in the Western Pacific Region: Successes and challenges. International Health. 2021; 13: S10âS16. Publisher Full Text\n\nYou Y, Lai X, Pan Y, et al.: Artificial intelligence in cancer target identification and drug discovery. Signal Transduction and Targeted Therapy. 2022; 7(1): 1â24. Publisher Full Text\n\nZeynudin A, Degefa T, Tesfaye M, et al.: Prevalence and intensity of soil-Transmitted helminth infections and associated risk factors among household heads living in the peri-urban areas of Jimma town, Oromia, Ethiopia: A community-based cross-sectional study. PLoS One. 2022; 17(9 September): 1â17.\n\nZhang Q, Yang L, Zhou F: Attention enhanced long short-term memory network with multi-source heterogeneous information fusion: An application to BGI Genomics. Information Sciences. 2021; 553: 305â330. Publisher Full Text\n\nZhou G, Chen M, Ju CJT, et al.: Mutation effect estimation on proteinâprotein interactions using deep contextualized representation learning. NAR Genomics and Bioinformatics. 2020; 2(2): 1â12. Publisher Full Text"
}
|
[
{
"id": "208370",
"date": "10 Oct 2023",
"name": "Anupam Nath Jha",
"expertise": [
"Reviewer Expertise Computational Biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have written about different NTDs and machine learning techniques in the review article.\nDifferent types of research works are done and reviewed on neglected tropical diseases (e.g. Jha, 20231). Here the authors have listed all such diseases and explain the causing agent. Further the application of advance machine learning approaches in drug discovery and development of such diseases are elaborated.\nIt is good to read articles about the present status of NTDs but the authors are suggested to add the future scope of different computational approaches in surveillance, management and treatment of these diseases. Also, the limitations of people working in this field should be mentioned because limited data is not the only problem in handling NTDs.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": []
},
{
"id": "215469",
"date": "15 May 2024",
"name": "Erma Sulistyaningsih",
"expertise": [
"Reviewer Expertise Molecular parasitology",
"molecular medicine"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI found some references are more than 5-years old publications (2015, 2012, 2013, 2016).\nThe manuscript is about the application of machine learning for Neglected Tropical Diseases (NTDs), the authors already mentioned the list of 20 diseases of NTDs recognized by WHO (Table 1), however, the authors also write on malaria in the next session. Please specify the discussion on the 20 NTDs.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "11629",
"date": "28 Jun 2024",
"name": "Ethan Khew",
"role": "Author Response",
"response": "Dear Erma Sulistyaningsih, Thank you very much for your valuable feedback on our manuscript. We appreciate the time and effort you have taken to review our work and provide constructive comments. Regarding the use of references older than five years, we selected these sources intentionally as many of them are seminal works or original studies that first addressed key issues (e.g., global distribution burden studies). These foundational papers are frequently cited by more recent publications, and we believe it is more ethical and academically honest to acknowledge the original sources. However, we understand the importance of including up-to-date references, and we will review our citations to ensure a balanced representation of both seminal and recent works. We will also add perspectives from recent publications, but we have noted that there is a lack of new research or evaluation on certain topics (such as disease burden), leading us to rely on older, yet still relevant, publications. Concerning the inclusion of malaria in our discussion, we recognize that this might appear to deviate from the list of 20 NTDs recognized by the WHO. Our review is specifically focused on the Southeast Asia and Western Pacific regions, where there is a limited number of machine learning-based studies on the recognized NTDs. To provide a comprehensive overview, we expanded our scope to include other significant diseases such as malaria and melioidosis, which are of considerable concern in these regions. Both diseases, we believe, merit attention and potential inclusion in the NTD list due to their impact. We apologize for any confusion this may have caused and are open to amending the manuscript to clarify our rationale and align more closely with the recognized NTDs if that would address the concern. We hope this clarifies our decisions and are ready to make necessary adjustments to improve the manuscript once we hear back from you. Thank you once again for your insightful feedback. Best regards, Ethan Khew"
}
]
}
] | 1
|
https://f1000research.com/articles/12-287
|
https://f1000research.com/articles/12-285/v1
|
14 Mar 23
|
{
"type": "Brief Report",
"title": "Prevalence of oral HPV among people living with HIV (PLHIV) in Pune, India",
"authors": [
"Ivan Marbaniang",
"Samir Joshi",
"Rohidas Borse",
"Samir Khaire",
"Rahul Thakur",
"Prasad Deshpande",
"Vandana Kulkarni",
"Amol Chavan",
"Smita Nimkar",
"Vidya Mave",
"Samir Joshi",
"Rohidas Borse",
"Samir Khaire",
"Rahul Thakur",
"Prasad Deshpande",
"Vandana Kulkarni",
"Amol Chavan",
"Smita Nimkar",
"Vidya Mave"
],
"abstract": "Background: People living with HIV (PLHIV) are at an increased risk of human papillomavirus (HPV)-related head and neck cancers (HNCs). However, there is little data on the prevalence of oral HPV among PLHIV in India, limiting the planning of oral HPV preventive strategies. Methods: We used cross-sectional data from an oral cancer screening study conducted at the antiretroviral therapy (ART) centre of Byramjee-Jeejeebhoy Government Medical College-Sassoon General Hospitals (BJGMC-SGH). PLHIV â¥21 years of age with no prior history of HNCs were enrolled. We determined the prevalence of high-risk oncogenic HPV (hrHPV) and low-risk non-oncogenic HPV (lrHPV) using real-time PCR and Next-Generation Sequencing. We used multinomial logistic regression to determine the prevalence ratios (PRs) of different sociodemographic, clinical, and behavioural predictors with hrHPV and lrHPV. Multivariable models were adjusted for age, sex, CD4 count and duration on ART. Results: Of the 582 PLHIV enrolled, the median age was 40 years (IQR: 34â46) and 54% were male. More than a fourth (25.8%) had multiple sexual partners and 11% had given oral sex. Median CD4 counts were 510 cells/mm3 (IQR: 338â700). The prevalence of hrHPV was 4.5% and lrHPV was 3.4%. Of those with hrHPV, 77% had HPV16. There were no significant associations with any predictors for both lrHPV and hrHPV in adjusted analyses. Conclusions: We found the prevalence of any oral HPV (hrHPV and lrHPV) to be 7.9% among PLHIV in India. Larger studies are required to better understand risk factors for oral HPV among Indian PLHIV.",
"keywords": [
"oral HPV",
"HIV",
"India"
],
"content": "Introduction\n\nPeople living with HIV (PLHIV) have a 2â3 times higher odds of oral human papillomavirus (HPV) prevalence compared to HIV-uninfected individuals.1 This is estimated to increase their risk of HPV-associated head and neck cancers (HNCs) 1.2â2.1 times relative to HIV-uninfected individuals.2 However, estimates of oral HPV prevalence among PLHIV are mostly derived from high income countries (HICs) and data from low- and middle-income countries (LMICs) remain scarce, despite the burden of HIV in LMICs being considerably higher than HICs.3\n\nGlobally, India has the third highest number of PLHIV (approximately 2.3 million).4 Only one study from the country has previously reported on oral HPV prevalence among PLHIV, restricted to a key population, men who have sex with men (MSM).5 To develop comprehensive strategies for oral HPV prevention that are geographically relevant for PLHIV, it is also important to report on the prevalence of oral HPV among PLHIV that are not key populations. In this manuscript, we estimate the prevalence of oral HPV and assess associated factors among PLHIV in Pune, India.\n\n\nMethods\n\nThe Ethics Committee of Byramjee Jeejeebhoy Government Medical College â Sassoon General Hospitals (BJGMC-SGH) (Date of approval: 19 September 2016) and the Institutional Review Board of Johns Hopkins University (IRB00118708, Date of approval: 13 December 2016) approved the study. It should be noted that the Ethics Committee of BJGMC does not issue ethical approval numbers, nor does it have an Ethics Number itself.\n\nWe used data from a parent oral cancer screening study conducted between June 2017 and June 2019 at Byramjee Jeejeebhoy Government Medical College â Sassoon General Hospitals (BJGMC-SGH), a tertiary health care centre in Pune, India.6 The parent study enrolled 1,234 participants, including 633 HIV-uninfected individuals and 601 PLHIV. For this analysis, data from HIV-uninfected participants and PLHIV with missing data for oral HPV (n=19) were excluded. Details of the study including enrolment procedures are provided elsewhere.6 This analysis included data of PLHIVâ¥21 years of age attending the ART centre for care, with no prior history of HNCs who provided a 30 seconds oral rinse and gargle sample, and written informed consent.\n\nSociodemographic (age, biological sex), behavioural factors (smoking, smokeless tobacco (SLT) and alcohol use), sexual history (men who have sex with men (MSM), oral sex, lifetime sexual partners), clinical history (CD4 counts, duration on antiretroviral therapy, suspected oral potentially malignant disorder (OPMD), HPV results including genotype) data were extracted from the database of the parent study.6 These data were collected by trained non-medical health care professionals of the study team in the parent study.\n\nAssuming that the prevalence of oral HPV was 24%, consistent with the previous study that reported on oral HPV prevalence among Indian MSM living with HIV,5 our sample size of 582 was >95% powered at an alpha of 0.05.\n\nThe parent study6 used polymerase chain reaction (PCR) and next-generation sequencing (NGS) to obtain the prevalence of oral HPV. PCR and NGS were performed at a private laboratory (GenePath Diagnostics, Pune). These procedures are described below.\n\nReagents\n\nPlatinum Taq DNA polymerase (Invitrogen, Cat No. 10966034), HotStarTaq DNA polymerase (Qiagen, Cat No. 203205), dNTP (deoxyribonucleotide triphosphate) 25 micromolar (mM) (Lucigen, Cat No. D59104) were used. [Note - Invitrogen PCR buffer and MgCl2 are provided by Invitrogen along with Platinum Taq DNA polymerase].\n\nThermal cycling conditions, primers used and model of PCR machine\n\nA nested PCR approach targeting the L1 gene was used. The outer PCR was carried out using an equimolar mixture of each PGMY09 and PGMY11 primer pool7 at a concentration of 80 nanomolar (nM) each, which generated a 450 base pairs (bp) product. This product was used as a template for the inner PCR. The inner PCR was carried out using custom tagged GP5+ and GP6+ primers7 at a final concentration 200 nM each, which generated a 150 bp product. The tagged inner PCR products were used for library generation where specific barcodes were incorporated for each sample. The library was sequenced on an Illumina MiSeq or a NextSeq next generation sequencer in a shared run using the Illumina V2 2*250 (MiSeq) or Mid-output 2*150 (NextSeq) paired-end sequencing by synthesis chemistry. The resulting .fastq raw read files were aligned against HPV DNA database using in-house bioinformatics pipeline and read counts generated were analysed for presence/absence and type of HPV DNA in each sample.\n\nThe outer PCR was carried out at 20 microlitre (ÎŒl) reaction volume, with 1X Invitrogen PCR buffer, 4 mM MgCl2, 0.2 mM dNTP and a blend of two polymerases, 0.5U of Platinum Taq DNA polymerase (Invitrogen) and 0.5U of HotStarTaq DNA polymerase (Qiagen). The outer PCR was carried out on Veriti 96-well thermal cycler (Applied Biosystems) with initial incubation at 37°C, initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 95°C for 20 sec, annealing at 56.5°C for 30 sec, extension at 72°C for 1 min. The amplified outer PCR products were diluted 100 times with ultrapure nuclease free PCR grade water and used as a template for inner PCR.\n\nFor the inner PCR, custom tagged GP5+ and GP6+ primers7 were used at a final concentration of 200 nM each. The inner PCR was carried out at 20 ÎŒl reaction volume with 1X Invitrogen PCR buffer, 4 mM MgCl2, 0.2 mM dNTP, 0.5 mg/ml BSA, and a blend of two polymerases, 0.5U of Platinum Taq DNA polymerase (Invitrogen) and 0.5U of HotStarTaq DNA polymerase (Qiagen). The inner PCR was carried out on Veriti 96-well thermal cycler (Applied Biosystems) with initial incubation at 37°C, initial denaturation at 95°C for 15 min, followed by eight cycles of denaturation at 95°C for 20 sec, ramp up annealing from 40°C to 72°C for 30 sec, extension at 72°C for 30 sec, followed by 27 cycles of denaturation at 95°C for 20 sec and extension at 72°C for 1 min.\n\nPCR (and NGS) results were treated as the outcome, classified into three groups: 1) no HPV; 2) high risk or oncogenic HPV (hrHPV i.e., if they belonged to any of the following genotypes HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68)8; and 3) low risk HPV (6/11, 7, 13, 27, 32/42, 34, 43/91, 44, 53, 62, 71, 72, 73, 81, 82, 83, 84, 86/87, 89/102, 90/106, 107, 114, and 120).8\n\nMedians and proportions across the outcome were compared using Kruskal Wallis and Fisherâs exact tests, respectively, for different sociodemographic, behavioural, sexual history and clinical predictors (Table 1). We used multinomial logistic regression to obtain prevalence ratios (PRs) comparing lrHPV and hrHPV groups (comparison groups) against the no HPV group (reference group) for different predictors. Multivariable models were adjusted for variables chosen a priori as predictors of oral HPV and that had a minimum of five participants when cross tabulated against the outcome. These were age, sex, CD4 counts and duration on ART.\n\n* There are no significant differences between the medians or proportions across no HPV, hrHPV (high risk HPV) and lrHPV (low risk HPV) groups (p-value associated with Kruskal- Wallis and Fisherâs exact test >0.10 for all characteristics.\n\na SLT: Smokeless tobacco, which are forms of tobacco that are not burned. Locally available forms available: khaini (tobacco + slaked lime); gutka (tobacco + areca nut + slaked lime); mishri (roasted powdered tobacco); paan (tobacco + areca nut + slaked lime + condiments, wrapped in a betel leaf); paan masala; snuff.\n\nb MSM: Men who have sex with men.\n\nc ART: Antiretroviral therapy.\n\nd Regression estimates are interpreted with respect to an increase in 50 CD4 cell counts; CD4 count values were missing for 8 participants.\n\ne OPMD: Oral potentially malignant disorder.\n\nWe performed three sensitivity analyses, comparing primary results with: 1) Findings in which CD4 counts were categorized into <200 and â¥200 cells/mm; 2) Findings from models in which PRs were obtained for a binary outcome i.e., no HPV and any HPV (collapsing hrHPV and lrHPV into a single group); 3) Findings from regression models in which imputations using chained equations for those with missing HPV were performed.\n\nStatistical significance was set to a two-sided p-value of 0.05. All analyses were performed in Stata 17.0 (RRID:SCR_012763) (free alternative, RStudio).\n\n\nResults\n\nA total of 582 PLHIV were enrolled. The median age was 40 years (IQR: 34â46), 46.4% (n=270) were biologically female, 18.2% (n=106) had smoked, 41.1% (n=239) had used smokeless tobacco (SLT) and 35.2% (n=205) had consumed alcohol. Approximately 2.5% (n=14) self-identified as MSM, 10.5% (n=61) had given oral sex and 25.8% (n=150) had multiple sexual partners, in their lifetime. Median time updated CD4 counts were 510 cells/mm3 (IQR: 338â700) and median duration on ART was eight years (IQR: 4â12). None of the participants were vaccinated against HPV.\n\nThe prevalence of hrHPV was 4.5% (n=26) and lrHPV was 3.4% (n=20). Of those with hrHPV, 76.9% (n=20) had HPV16. Approximately 40% of those with hrHPV (n=8) and 50% (n=10) of those with lrHPV had never smoked, used SLT or consumed alcohol. The distribution of hrHPV was HPV16: n=20, HPV18: n=2, HPV35: n=1, HPV66: n=2 and HPV68: n=1; and that of lrHPV was HPV7: n=1, HPV13: n=1, HPV27: n=1, HPV32: n=1, HPV42: n=1, HPV44: n=3, HPV72: n=3, HPV81: n=1, HPV84: n=1, HPV90: n=1, HPV107: n=4, HPV120: n=2 (Table 1).\n\nThe prevalence of hrHPV and lrHPV were not statistically significantly different (i.e., p>0.05) by age, biological sex, tobacco and alcohol use habits, and sexual and clinical history.\n\nIn the univariate analysis, relative to the no HPV group, a unit increase in age was associated with 5% increase in the prevalence of lrHPV (PR: 1.05; 95% CI: 1.01, 1.10). There were no predictors significantly associated with hrHPV in univariate analysis. When adjusted for age, sex, time updated CD4 counts and ART duration, no predictors were significantly associated with either lrLPV or hrHPV (Table 1).\n\nSensitivity analyses findings were consistent with results presented in Table 1. However, when CD4 counts were categorized into <200 and â¥200 cells/mm3, participants with <200 cells/mm3 had a 3.14- (95% CI: 1.20, 8.22) and 3.12- (95% CI: 1.19, 8.20) fold higher prevalence of hrHPV in univariate and multivariable analyses, respectively, but associations with lrHPV were non-significant. We do not present these as primary findings because there were no female participants with CD4 counts <200 cells/mm3 in the hrHPV group and multivariable results are likely an extrapolation by the model.\n\n\nDiscussion\n\nUsing oral rinse samples, we found the overall prevalence of any HPV to be 7.6%, lrHPV to be 3.4% and hrHPV to be 4.5% among PLHIV. To our knowledge this is the first study from India to report on oral HPV among PLHIV that are not exclusively MSM and the largest study from the country to date to report on the prevalence of oral HPV.9,10\n\nWe found combined oral HPV (i.e., any oral HPV: lrHPV and hrHPV) and hrHPV (only) prevalence to be lower than what has been reported among studies in PLHIV from the United States.1 However, the sample size of the present study is larger than most of these studies. Compared to the study conducted among MSM living with HIV in India, our overall prevalence of any oral HPV was lower (7.6% versus 23.7%), hrHPV prevalence was higher (4.5% versus 2.4%) and hrHPV was almost exclusively HPV16 (77% versus 0%).5 These findings indicate the heterogeneity of oral HPV prevalence and genotypes among different subgroups of PLHIV in India and the need for larger studies to better characterize oral HPV in them.\n\nMost previous studies in India have only reported on oral HPV among HIV-uninfected individuals with HNCs. The prevalence of oral HPV in these studies varies between 0â79%, with HPV16 being the most common genotype.9,10 More than three-fourths of those with hrHPV in our study had HPV16. Further, 40% of those with hrHPV reported to have never smoked, used SLT or consumed alcohol, which are traditional risk factors for HNCs. Oral HPV screening is not routinely performed as part of HIV care at present. Given the strong association between HPV16 and HNCs,1 and the increased vulnerability of PLHIV for HNCs,1 oral HPV assessment for PLHIV must be considered.\n\nWe found no association with CD4 counts in our primary analysis. This may be due to the high median CD4 counts of the study population. Immunosuppression is an important risk factor for oral HPV,1,8 and our sensitivity analysis indicates that those with CD4 counts <200 cells/mm3 may be more likely to have prevalent hrHPV. A previous publication from our site showed that a large proportion of PLHIV consistently presented with CD4 counts <200 cells/mm3 over a 12-year period (2005â2017).11 This underlines the need to facilitate earlier engagement of PLHIV in HIV care, to reduce their risk of oral HPV acquisition.\n\nThere are several limitations in our study that merit discussion. We did not measure HIV viral loads and are unable to comment on the association between viral suppression and oral HPV prevalence. We did not find associations between oral HPV with either multiple sexual partners or oral sex. This is likely due to participants underreporting these behaviours, driven by social desirability. We are committed to improving on this aspect and have organized a series of workshops for sexual history assessment. While this is the largest study till date to report on oral HPV from India, we were not powered to detect significant associations especially in multivariable models. Our findings may also not be generalizable to other PLHIV living elsewhere in India.\n\nPLHIV are a vulnerable group for HPV-associated HNCs and those living in LMICs may be particularly at risk because oral HPV screening is not routinely performed as part of HIV care. We reiterate the need for larger studies to better understand oral HPV prevalence and associated risk factors among PLHIV in India, simultaneously with the scaling-up of oral HPV screening measures among them.",
"appendix": "Data availability\n\nData cannot be shared publicly because it comes from people living with HIV in India, many of whom have not disclosed their HIV status except to their HIV health care provider and spouses. Data are available from the Ethics Committee - Byramjee Jeejeebhoy Medical College & Sassoon General Hospitals, Pune, India (contact via email @bjmcecirb@gmail.com) for researchers who meet the criteria for access to confidential data.\n\n\nAcknowledgements\n\nWe would like to thank the non-medical health care workers involved: Suhasini Surwase, Archana Pawar and Kanta Zarekar; the data entry operator: Rohini Kamble; all the ART staff and the study participants without whom this work would not be possible.\n\n\nReferences\n\nBeachler DC, D'Souza G: Oral human papillomavirus infection and head and neck cancers in HIV-infected individuals. Curr. Opin. Oncol. 2013; 25(5): 503â510. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChaturvedi AK, Madeleine MM, Biggar RJ, et al.: Risk of human papillomavirus-associated cancers among persons with AIDS. J. Natl. Cancer Inst. 2009; 101(16): 1120â1130. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShao Y, Williamson C: The HIV-1 epidemic: low- to middle-income countries. Cold Spring Harb. Perspect. Med. 2012; 2(3): a007187. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPress Trust of India: India has 3rd-highest number of HIV-infected people: UN. The Hindu; 2014. Accessed August 15, 2022. Reference Source\n\nHernandez AL, Karthik R, Sivasubramanian M, et al.: Prevalence of oral human papillomavirus infection among Indian HIV-positive men who have sex with men: a cross-sectional study. BMC Infect. Dis. 2021; 21(1): 675. [published Online First: 20210712]. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarbaniang I, Joshi S, Sangle S, et al.: Smokeless tobacco use and oral potentially malignant disorders among people living with HIV (PLHIV) in Pune, India: Implications for oral cancer screening in PLHIV. PLoS One. 2022; 17(7): e0270876. [published Online First: 20220705]. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGravitt PE, Peyton CL, Alessi TQ, et al.: Improved amplification of genital human papillomaviruses. J. Clin. Microbiol. 2000; 38(1): 357â361. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuller K, Kazimiroff J, Fatahzadeh M, et al.: Oral Human Papillomavirus Infection and Oral Lesions in HIV-Positive and HIV-Negative Dental Patients. J. Infect. Dis. 2015; 212(5): 760â768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNair D, Mair M, Singh A, et al.: Prevalence and Impact of Human Papillomavirus on Head and Neck Cancers: Review of Indian Studies. Indian J. Surg. Oncol. 2018; 9(4): 568â575. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBruni L, Albero G, Serrano B, et al.: Human Papillomavirus and Related Diseases in India. ICO/IARC Information Centre on HPV and Cancer (HPV Information Centre). Summary Report 22 October 2021. Accessed August 5, 2022. Reference Source\n\nRaichur P, Salvi SP, Sangle S, et al.: Trends and Sex Differences in Access to HIV Care with Scale Up of National HIV Treatment Guidelines in Pune, India. J. Int. Assoc. Provid. AIDS Care. 2020; 19: 2325958220931735. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "180179",
"date": "04 Sep 2023",
"name": "Sneha Sethi",
"expertise": [
"Reviewer Expertise Oral cancers",
"HPV",
"Minority populations",
"racism"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReviewer comments: This is very important and highly significant study, very few studies report on this prevalence although very common in PHIV and should be reported more. Although, I would suggest some major revisions before proceeding towards submission and publication.\nIntroduction: The introduction is very brief, I would suggest adding another section in the beginning related to global statistics of Oral HPV associated HNCâs in PHIV populations, also describing high and low risk HPV infection and also a reference of why PHIV would be more susceptible for this infection. This will make the introduction more engaging and informative for a wider audience instead of people from a very specialized niche.\nMethods: I would like to applaud the efforts of the author team on compiling the methods and seeking proper ethical approval from both institutions before commencing the study.\nResult and discussion are satisfactory with minor grammatical errors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "246090",
"date": "01 Mar 2024",
"name": "Bianca Da Costa Dias",
"expertise": [
"Reviewer Expertise HPV",
"STIs"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article provides the prevalence of oral HPV amongst HIV infected individuals on ART in Pune India and is described as the largest study of this nature to date. However, the authors fail to identify any associated risk factors and concede that larger studies are required to establish risk factors for oral HPV in PLHIV in India.\nOverall, the article is well written, and the data presented is of importance. Throughout the manuscript however I feel it is important to emphasise that this cohort is PLHIV on ART- and may thus differ substantially from PLHIV who are not on ART.\nIntroduction: The manuscript would benefit from data regarding the Prevalence/ Incidence of Head and Neck Cancers in India as stratified by HIV status (if available)- to put this study into context.\n\nMethods: Details regarding the processing of oral rinse samples is lacking - the volume of sample collected per patient, how these were treated prior to DNA extraction etc.\n\nThe authors base their sample size estimates on a prevalence of 24%, however, the actual observed prevalence of HPV is much lower. How did this affect the power of the study? This should be commented on in the discussion.\n\nThe heading \"Thermal cycling conditions....PCR machine\" should be changed to PCR amplification and Sequencing. In this section, the paragraphs detailing the nested PCR procedures (outer and inner PCR) should be moved prior to NGS methods. Could the choice of method- PCR amplification using PGMY primers have introduced bias based on the preferential amplification of most abundant genotypes? Perhaps this can be briefly commented on if the authors feel it appropriate.\n\nSince data was generated for individual HPV genotypes- these should be available as supplementary tables.\n\nThe authors discuss \"any HPV\" results, but these are not presented in the tables. This should be included. The data could also be analysed to assess number of multiple HPVs/ co-infections and if this variable is maybe associated with specific risk factors.\n\nAlthough the variables are those usually included in such studies- the lack of quantification is a limitation that the authors must mention. Often it is not just having had multiple lifetime partners but the actual NUMBER of lifetime vaginal/oral sex partners or number of smoked packs etc that are associated with oral HPV risk. Furthermore, some of these variables could be past vs current use (smoking, alcohol use). Perhaps stratification in this way may allow for differences to be observed. If not, and these were already considered, then the authors should mention this.\n\nTable 1: The denominator of MSM should be men only (N=312) and not all participants. Furthermore, the high prevalence of OPMD, particularly amongst those with NO detectable HPV warrants discussion.\n\nDiscussion: The authors fail to provide a rationale for the low prevalence of HPV in this cohort- especially when compared to the HIV uninfected prevalence they cite from literature. This sub-study explicitly excluded the HIV uninfected participants enrolled in the larger study as mentioned in the methods. This would have been a better comparator group to put these findings into context and to advocate for the screening recommended in the concluding paragraph as the low prevalence of HR-HPV does not support this recommendation (in my opinion).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "246084",
"date": "05 Mar 2024",
"name": "Tesfaye Gelanew",
"expertise": [
"Reviewer Expertise Viral Immunologist"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study on oral HPV prevalence among people living with HIV (PLHIV) in Pune, India, where there is currently a dearth of data, provides valuable insights, but indeed, there are several points of contention that warrant further attention by authors:\nRisk Factors for Oral HPV in PLHIV:\nThere is ongoing debate about the risk factors for oral HPV infection in PLHIV. Multiple studies indicate that HPV prevalence (both low-risk and carcinogenic types) is higher in PLHIV compared to the HIV-negative population. However, the specific risk factors contributing to the high prevalence of HPV infection in PLHIV were identified in the present study.\n\nLow HPV Prevalence and ART:\nThe observed low prevalence of oral HPV in PLHIV might be attributed to participants being on antiretroviral therapy (ART) for an extended period. ART could potentially lead to clearance of HPV infection, but the current study does not directly address or support this hypothesis.\n\nAge Group Selection:\nThe authors chose to focus on PLHIV aged 21 years and older. I'm not sure why the authors chose to focus on PLHIV who were 21 years of age and older. It would have been relevant to investigate rates of persistent HPV infection and precancerous lesions across different age groups. Understanding how HPV behaves over time in PLHIV of varying ages could provide valuable insights.\n\nLow HPV prevalence and HPV Vaccination:\nBeing vaccinated could account for reduced oral HPV prevalence in PLHIV of the present study. Unfortunately, the study does not report whether participants had received the HPV vaccine. Given the rollout of the HPV vaccine in India, this information would have been crucial in interpreting the observed low prevalence.\n\nInclusion and Exclusion Criteria:\nThe study lacks clarity regarding its inclusion and exclusion criteria. Ambiguities in these criteria may contribute to the inconsistent results reported in the study.\n\nIn summary, while the study shed light on oral HPV prevalence in PLHIV, addressing the aforementioned points could enhance the quality of the paper and our understanding of HPV dynamics in this population and geography.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-285
|
https://f1000research.com/articles/12-284/v1
|
14 Mar 23
|
{
"type": "Study Protocol",
"title": "Evaluation of serum VEGF-A and interleukin 6 as predictors of angiogenesis during peri-implantation period assessed by Transvaginal Doppler Ultrasonography amongst women of prior reproductive failure: a cross-sectional analytical study.",
"authors": [
"Priya Pratapan Nair",
"Deepti Shrivastava",
"Deepti Shrivastava"
],
"abstract": "Background: Infertility is a wide spectrum of disorder affecting many couples and is defined as the inability of a couple to achieve pregnancy. A series of investigations are required to know the reason behind infertility, yet a small percentage (08-37%) of couples exist, in which no obvious cause is delineated. Different hormones, growth factors and interleukins are responsible for successful ovulation, implantation and further growth of embryo. Endometrial receptivity is an important predictor of the outcome of implantation and further growth of embryo. Methods: The study would be conducted in a tertiary care centre over a period of two years from July 2022 till July 2024 on non-pregnant unexplained infertile women (63) who will undergo In vitro Fertilisation and Embryo Transfer (IVF-ET) in the following cycle and the result will be compared with the fertile women (21) with same demographic features. The levels of serum Vascular Endothelial Growth factorâA, interleukin-6 will be measured on day 21 of the menstrual cycle in both the group, simultaneously, transvaginal ultrasonography and doppler findings will be measured in both groups to see for endometrial receptivity. The values will be compared for any significance. A new Scoring system will be generated of these three parameters PREDICGIO scoring system after pilot study on ten patients. The values of Serum VEGF-A, IL6 and Doppler findings will be compared between fertile and infertile group to see for significant difference. Conclusion: The current study helps in determining the suboptimal vasculogenesis and angiogenesis in women with failure in reproduction. It will also help to a great extent in managing patients of reproductive failure with immunomodulator drugs thus providing external angiogenic factors to help in conceiving and carrying pregnancy till term.",
"keywords": [
"Infertility",
"growth factors",
"hormones",
"interleukins",
"VEGF-A",
"IL6",
"Transvaginal Ultrasonography",
"Doppler",
"IVF-ET."
],
"content": "Introduction\n\nInfertility is a wide spectrum of disorders affecting many couples and is defined as the inability of a couple to achieve pregnancy even after one year of marriage and not using any contraception, in spite of all the conventional investigations of both partners, women in reproductive age group between the 15-49 age group. A series of investigations are required to know the reason behind infertility, yet a small percentage (8-37%) of couples exist, in which no obvious cause is delineated.1\n\nInfertility can be primary where the couple has not yet achieved pregnancy or secondary infertility where the couple who have been able to get pregnant at least once, but now are unable.2 WHO stated that most of the patients suffer from primary infertility. Currently, the prevalence of infertility among the Indian population is 17.9%, (NFHS-IV), higher in urban areas.2 WHO has stated the prevalence of primary infertility between 3.9% to 16.8% in India.2\n\nInfertility causes enormous emotional, physiological, psychological, sexual, social and financial burden on family. Infertility is a relative process.3 The process is complex in both men and women. About 8-10% of couples are affected by infertility.4 Evaluation of infertility requires simultaneous counselling of both couples to be time saving and simultaneous treatment of either partner can be started.\n\nSuccessful pregnancy is characterised by many factors including cervical, tubal, ovarian, uterine, peritoneal and male factors. Even after having no abnormalities in any of these factors, couples have unexplained infertility. After ovulation occurs, the successful implantation depends on proper tubal motility with appropriate development of embryo to the blastocyst stage, mobility of embryo to the uterine cavity and requirement of a receptive endometrium for further implantation and growth of embryo.\n\nImplantation is a very intricate process which is governed by a number of growth factors, hormones and inhibitory and supportive cytokines. Combination and coordination of all the factors are required for implantation of the embryo on endometrial surface to achieve a successful pregnancy. The implantation process also requires coordinated effects of autocrine, paracrine and endocrinological factors.5\n\nEndometrial receptivity for a successful implantation is a series of events that take place at the embryo and endometrial junction which determines the outcome of pregnancy. Appropriate and adequate angiogenesis and vasculogenesis are required for successful implantation and development of embryo for successful growth of pregnancy.6 In the first few weeks of pregnancy (1-2 weeks), capillaries grow and covers the syncytiotrophoblastic lacunae. The stages of implantation take place in steps as follows: first apposition then adhesion followed by invasion of maternal decidua by the developing embryo. Initially, the capillaries surrounding the syncytiotrophoblast constitutes the vascular supply of the rapidly growing embryo.7 VEGF forms one of the most essential pro-angiogenic factor. It is responsible for the early placental vascular changes.8 It is produced by stromal and epithelial cells in the top layers of the uterine endometrial layers and the embryo and it is a soluble angiogenic factor. The receptors of VEGF are found in the endothelium which regulates various functions of endothelium. VEGF has mitogenic action on the microvasculature and macrovasculature of endothelium derived from lymphatics and blood vessels.9,10\n\nVEGF is responsible for physiological and pathological development of vessels.11 VEGF is amongst sub-category of growth factors. It is a glycoprotein which is homodimeric and of 45,000 Daltons. VEGF-A also called as VEGF and was the first of VEGF family to have been known. The VEGF class has five subtypes: VEGF-A, B, C, D and Placenta Growth Factor (PGF).10,12 Abnormalities in vascular endothelial growth factor can cause Utero-placental insufficiency as in cases of Growth Retardation in utero (IUGR), pre-eclampsia and in many cases of unexplained recurrent abortions.13\n\nRecurrent miscarriages or recurrent abortions are said when there are 2 or more continuous or recurrent pregnancy loss before 20 weeks of pregnancy or foetus weighing less than 500 grams from the date of last menstrual period as per American Society for Reproductive Medicine.14,15 It affects at least 2-4% of couples who are trying to conceive. Many factors like anomalies, endocrinological, autoimmune, infectious, thrombophilic and chromosomal abnormalities have been found to be some of the causes of recurrent abortions. In over 50% cases, the causes of recurrent abortions are unexplained.16,17\n\nReduced levels of pro vasculogenic factors like VEGF-A, C and their receptors on the endometrium has been suggested as cause of spontaneous abortions, as it mainly affects the foetal and placental angiogenesis. Women having infertility and recurrent miscarriages have been found to have low levels of VEGF.18,19\n\nAnother important factor postulated in infertility and recurrent abortions is serum Interleukin 6 (IL6). Interleukin 6 is vital in division and attachment of trophoblastic cells and is helpful in implantation and pregnancy.5,20 It comes under the Th2 immune response family and is shown to have an important effect on implantation, angiogenesis and pregnancy outcome. Decreased levels of IL6 in ovarian follicles cause increased chances of pregnancy in patients undergoing In vitro Fertilisation.21 Elevated IL6 was found in patients of unexplained infertility, recurrent abortions, preeclampsia and preterm deliveries.22\n\nTransvaginal ultrasonography with doppler flow plays an essential role in infertility and recurrent abortion management. Ultrasonography helps in determining uterine abnormalities, cervical abnormalities, tubal anatomy, ovarian reserve and peritoneal adhesions. During perimenstrual period and during implantation window, it helps to determine the angiogenesis and vasculogenesis by assessing the sub endometrial blood flow, zones of endometrium, resistance index (RI) and pulsatality index (PI) of the uterine arteries. It can aid us in the cases of infertility and recurrent miscarriages so as to provide proper angiogenic factors to the suboptimal endometrium for better pregnancy outcome. Assessment of uterine and ovarian blood flow is important aspect of reproduction. Pulse doppler and colour doppler helps in determining uterine and ovarian blood flow which changes dynamically according to the hormonal changes during menses. Doppler study helps to determine the sub endometrial blood flow and helps in accessing uterine receptivity.23\n\nIn the secretory phase, because of increased mucus and glycogen content within the glands of endometrium, the endometrium achieves a width between 8 and 16 mm and becomes echogenic with tortuous gland opening and tortuous vessels. The endometrium on an average, achieves its greatest thickness in the mid secretory phase of a spontaneous cycle, which measures up to 14 mm in width. Endometrial and sub endometrial blood flow measurements act as indicators of uterine receptivity and outcome of treatment.24,25\n\nIn the current study, reproductive failure will involve combination of both group of patients one who are dealing with failure to conceive conventionally or by various Artificial Reproductive Techniques used or those women who have conceived but could not carry pregnancy beyond first trimester.\n\nAs several studies are being conducted to expand the knowledge on various factors and hormones on reproduction and newer advanced technologies are being used, the success rate of pregnancy after various procedures of ART remains 40-50%. Still a 50-60% remains unexplained even after various determination of causes. Still a lot needs to be done to understand the pathophysiology behind actual cause of reproductive failure.\n\nCertain studies have shown that VEGF levels are decreased in patients with reproductive failure but some contradictory findings are observed in various other studies mentioned in review of literature below. Similar findings are observed in the levels of Interleukin- 6, some studies found no correlation of IL6 with reproductive failure while some studies found IL-6 levels lower in patients with reproductive failure.\n\nThe current study will throw light on the combined effect of growth factor VEGF, cytokine IL6 and transvaginal ultrasound with doppler in the concept of angiogenesis on reproductive failure, and differences in their levels with normal fertile non pregnant women, which will help in determining the suboptimal vasculogenesis and angiogenesis in women with failure in reproduction. It will also help to a great extent in managing patients of reproductive failure with immunomodulator drugs thus providing external angiogenic factors to help in conceiving and carrying pregnancy till term.\n\n\nProtocol\n\nThe study would be conducted on non-pregnant women attending the Outpatient Department or admitted ward patients in the Gynaecology and Obstetrics department of Datta Meghe Institute of Higher Education and Research and Jawahar Lal Nehru Medical College, Sawangi, Wardha, over a period of two years.\n\nStudy design: Observational, Analytical Cross-Sectional, Hospital based study.\n\nSample size: Calculated using Epi Info statistical software.\n\nTwo-sided confidence interval = 95%\n\nPower (percentage chance of detecting): 80%\n\nRatio (unexposed: exposed): 0.33\n\nPercentage outcome in unexposed group: 50%\n\nLeast extreme Risk ratio to be detected: 0.31\n\nLeast extreme Odds ratio to be detected: 0.18343\n\nPercentage outcome in exposed group: 15.5%\n\nN = 84 (63: exposed and 21: unexposed)\n\nInclusion criteria:\n\n1. Non pregnant women of age group between 20-40 years.\n\n2. Primary or secondary infertility patients having unexplained infertility with previous stimulated or unstimulated cycles.\n\n3. Patients with history of one or more abortions.\n\n4. Willing to give consent.\n\nExclusion criteria:\n\n1. Patients not giving consent.\n\n2. Patients with anatomical (uterine, tubal or cervical abnormalities diagnosed with ultrasonography or hysterosalpingography).\n\n3. Patients with chromosomal abnormalities diagnosed by chromosomal study will be excluded.\n\n4. Patients with autoimmune or endocrinological disorders diagnosed by blood tests or clinical features.\n\n5. Patients with chronic illness or infective aetiology which could be a cause of infertility or abortion.\n\nAim of the study is to evaluate levels of serum Vascular Endothelial Growth Factor-A and Interleukin 6 during periimplantaion period as predictors of angiogenesis by tvs in patients of reproductive failjure and compare levels with normal fertile women.\n\n\n\n1. To assess IL6 and VEGF-A serum level in relation to angiogenesis by Transvaginal ultrasonography in Reproductive failure group.\n\n2. To assess IL6 and VEGF-A serum levels correlation with angiogenesis by Transvaginal ultrasonography in fertile control group.\n\n3. Comparison of IL6 VEGF-A and angiogenesis in between women of fertile group and women of reproductive failure group.\n\n4. To assess the reproductive outcome of the women of study group in the same cycle.\n\n5. To assess the potential of combined values of VEGF-A, IL6 and Transvaginal doppler ultrasonography as a marker for endometrial receptivity.\n\n6. To compare the new scoring system generated out of 3 parameters (Serum VEGF-A, IL6 and TVS Doppler â PREDICGIO) with ERA on the basis of existing evidences in literature.\n\nControls will be of same demographic characters, non-pregnant women attending OPD of Obstetrics and Gynaecology for contraception or for gynaecological treatment other than that of fertility, during the same menstrual phase, who have at least one full term live child with no previous history of any abortions, still births or intra uterine demise and no other significant medical, anatomical, chromosomal, autoimmune, endocrinological or infective history in the past will be consented for their participation in research study and will be explained regarding the benefits of study to the population. They will be called during the implantation window phase for the collection of samples for VEGF-A and IL6 serum level estimation and their transvaginal ultrasonography will also be performed on the same day to assess the angiogenesis visually.\n\nEthical consideration: Ethical Committee Clearance: Datta Meghe Institute of Medical Sciences, Sawangi, Wardha, Institutional Ethical Committee clearance obtained on 29/9/2021.\n\nRe- regd no: ECR/440/Inst/MH/2013/RR-2019\n\nRef. no: DMIMS (DU)/IEC/2021/548\n\nWritten informed consents of cases and controls will be taken in their known language, confidentiality, privacy will be maintained, not to disclose the identity of cases and controls. They will be given particular out patient No. for identification and recovery of data.\n\nEthical consideration is taken into priority and the patient identity will not be disclosed.\n\nAfter taking prior informed consent for their inclusion in research study and explaining them details of the purpose of research, cases will be taken up for the study and worked up. History will be taken according to the proforma which will include age of both partners, demographic history, social history, duration of marriage, use of contraception, menstrual history, details of obstetrics history including previous history of abortions, still births, live births and intra uterine demise, whether histopathology of the specimen/chromosomal study of abortus was performed in the past pregnancy, how was the pregnancy terminated, personal history, medical or surgical history including hysteroscopy or laparoscopy for infertility in past, sexual history and relevant family history.\n\nAll the cases will undergo In vitro Fertilisation, and Embryo Transfer by Antagonist protocol. Details regarding their previous course of treatment, medications and various modalities of Artificial Reproductive Techniques used, hysterolaproscopy in past, number of previous cycles of undergoing IVF and ET will be asked.\n\nAnthropometry and general examination will be done. Basic lab investigation and some special investigations for patients with prior reproductive failure like blood sugar Fasting/Post meal, HbA1c level, serum fasting insulin, serum prolactin, Serum freeT3,fT4, TSH (fasting sample), coagulation profile (Prothrombin Time (PT)/activated Partial Thromboplastin Time (aPTT)), Antiphospholipid antibodies: Lupus Anti-coagulant (LAC), anticardiolipin antibody (ACL), b2glycoprotein, vaginal swab for culture, sensitivity and anatomical study of uterine structure and adnexa by ultrasonography if not already done will be done. HSG and karyotyping of partners (if required) will be done. Serum AMH, LH, FSH and Sr. oestradiol will be subjected to individual cases.\n\nPatients in both subtypes who will have any anatomic, infective, autoimmune, chromosomal or endocrinological abnormalities will be excluded from the study. Those participants with unexplained primary or secondary infertility with no known cause and history of abortion with no underlying factors will be taken up for further analysis in the research study.\n\nThe blood sample will be collected from cases of both group between day 21-23 of the menstrual cycle or during the day of Embryo Transfer in patients undergoing IVF-ET that is during peri implantation period for assessing the levels of serum VEGF-A and IL6 levels. The VEGF-A and IL 6 levels will be assessed through Enzyme-Linked Immuno sorbent assay (ELISA) technique -Human VEGF-A BIOLISA KIT and Human Interleukin 6 ELISA KIT. On the same day of sample collection, the cases will undergo transvaginal ultrasonography and doppler study to see for the endometrial thickness, pattern of endometrium, sub endometrial blood flow and the doppler indices of uterine arteries will be studied by using modified Applebaum scoring system which will help in visualising angiogenesis without invasive technique in the study group as well as control group.\n\n\nDiscussions\n\n\n\n1. A Study conducted in the Department of Immunology and Allergy, St Helier Hospital, U. K by Rhea Bansal, Brian Ford et al., titled Elevated Levels of Serum Vascular Endothelial Growth Factor-A Are Not Related to NK Cell Parameters in Recurrent IVF Failure. Background of the study was to establish relation between VEGF and NK cells in recurrent IVF failure patients as both the factors are responsible to play an important role in vasculogenesis and angiogenesis.\n\nIt was a case control study conducted in 62 women with repeated reproductive failure which was failed 3 attempts of IVF with fresh eggs, with 72 normal fertile women as control group. Women with autoimmune, endocrinological, anatomical factors associated with reproductive failure were excluded. Samples were collected in proliferative phase of menses and those women who underwent hormonal therapies were called for sample atleast after 2 months. The levels of VEGF-A, its receptor VEGF-R1, were estimated by ELISA kit and NK cells level by flow cytometry as anti-CD56, anti-CD16, anti-CD3 and anti- CD69 and NK cytotoxicity were determined.\n\nResult: The serum VEGF-A levels measured in 62 women with recurrent reproductive failure was significantly raised compared to the 72 healthy controls [median 362.9 pg/ml vs. 171.6 pg/ml). The serum soluble VEGF-R1 levels in the RIF group were similar to that of the control group. The median total NK count was 9.25%, the median NK CD69 count was 0.555Ã106/L and finally the median NK CD69 was 0.14Ã106/L. There was no correlation between these results and the corresponding VEGF-A and VEGF-R1 levels. The median killing of NK cells was 27.5% at 50:1, 19.5% at 25:1 and 12% at 12.5:1. There was no correlation between the NK cytotoxicity values at each effector: target ratio and serum VEGF-a and their receptor.\n\nConclusion of the study was that plasma VEGF levels were significantly raised in women with recurrent reproductive failure as compared to fertile women. No relation between VEGF levels and NK cells were found.26\n\n2. In a study conducted in the USA in the year 2014, in Obstetrics and Gynaecology department at Mercer School of Medicine, Central Georgia Fertility Institute, USA by Abdelmoneim Younis et al., the values of Interleukin 6, serum TNF-α, MCP-1 and paraoxonase-1 levels of women with recurrent pregnancy failure, endometriosis and PCOS were studied and evaluated.\n\nMethod: The study included thirty-six patients with Unexplained infertility, Endometriosis, PCOS who underwent controlled ovarian stimulation for either In vitro fertilisation or Intra uterine insemination. After their informed consent, serum sample were collected first on day 3 and again towards the end of treatment with Follicle Stimulating Hormone during (peak) and evaluated for the values of Interleukin-6, Tumour necrosis-α, MCP-1 and Paraoxonase-1.\n\nThe study group included thirty-six women with infertility (average of 33.7 ± 4.7 years within range of 26â44 years). The infertility women were further divided into three groups of infertility as: 1. Recurrent pregnancy failures (19.4%), 2. PCOS (38.9%) and 3. endometriosis (41.7%). All the women had normal puberty onset, sexual development and normal blood investigations. The patients were selected on the basis of normal blood investigations, usg pelvis, hormonal studies and husbandâs semen analysis. Unexplained infertility patients had all the normal profiles of both partners. Patients with cause of infertility as endometriosis underwent laparoscopy for classification. All the patients were given recombinant human follicle-stimulating hormone (rFSH) for controlled stimulation of ovaries. Injection was given once a day for 7â8 serial days and the development of follicules were monitored serially using serum E2 levels and TVS. An hCG injection was given 34 to 36 hours before the rupture of follicle to trigger maturation and rupture of dominant follicle. Patient was subjected to IVF or ICSI according to semen quality. Fertilized embryo was assessed for the cleavage and the embryos were transferred between day 3 and 5. Pregnancy was confirmed on day 14 of insemination using a positive serum β-hCG (>25 mIU/mL) or by transvaginal ultrasonography done at around 7 weeks after insemination to see the gestational sac for foetal pole and cardiac activity.\n\nResults obtained showed that the levels of IL6, MCP 1 and Paraoxonase-1 peaked positively along with peak of Estradiol levels. Mean of Tumour necrosis factor-α levels were statistically significant in Unexplained infertility group (P < 0.05). The serum levels of IL-6, and MCP-1 were significantly (P < 0.05) more in women with PCOS in contrast with that of Endometriosis and Unexplained infertility group. Tumour necrosis factor-α levels were negatively related to oestradiol levels and was less in pregnant women as compared to non-pregnant women (P < 0.05). There was no significant difference amongst the three groups when compared for patientâs age, FSH day 3 level, BMI, PON-1 and pregnancy outcome.\n\nConclusion generated out of the study was that the ovarian stimulation was the main factor for increased levels of circulating cytokines as seen by raised levels of PON-1, MCP-1 and IL-6 and reduced level of TNF-α after controlled stimulation of ovaries. Evidence of connection between mild endometriosis and cytokines were not established, PCOS was drawn to have relation with raised serum MCP-1 and IL-6 levels and lesser TNF-α level. No relationship could be found between PCOS and PON-1 level, mild endometriosis or recurrent pregnancy failure. Unexplained infertility was seen to have elevated TNF-α level.27\n\n3. Another study conducted in the Department of Obstetrics and Gynaecology, Uludag University School of Medicine, Turkey in 2016 by M. A. Atalay et al., showed clinical correlation of maternal serum vascular endothelial growth factor (VEGF) levels with idiopathic recurrent miscarriages.\n\nIn this study, 84 patients were sorted out in recurrent miscarriages group who had three or more spontaneous abortions and 47 women were identified with actual spontaneous miscarriages of which 25 women with idiopathic miscarriage were selected based on normal karyotyping, no infection, no autoimmune or hormonal imbalances. 25 women were followed up for study out of which 21 women conceived spontaneously who constituted the case group. They were compared with the control group which had 24 normally conceived women. Transvaginal obstetric ultra-sonographies were done and maternal sample collection weas done between 5th and 10th gestational weeks to compare serum VEGF and progesterone (P 4) levels. ELISA technique was used to measure VEGF and P4 levels.\n\nResults showed that the idiopathic cases amongst all the recurrent abortions were with 44.7% prevalance. Serum VEGF levels had no significant change when seen in the gestational age in the recurrent abortion and the control groups (P = 0.72 and P = 0.89, respectively). There was positive result between serum VEGF levels and the patientsâ age in recurrent abortions group (r = 0.515). Median of serum VEGF value was found higher in recurrent abortions group when compared to control group which was significant (210.33 ± 108.23 pg/ml vs.123.91 ± 18.8 pg/ml, respectively). There was no difference found in the idiopathic recurrent abortion group and control group in median P4 levels (19.53 ± 5.79 ng/ml and 20.08 ± 7.85 ng/m l, respectively). All different possibilities of recurrent abortions amongst patients (n = 22) were uterine anomalies (n = 5, 23%), autoimmune causes (n = 4, 18%), endocrine disorders (n = 4,18 %), hereditary thrombophilia (n = 7, 32%) and parental balanced translocations (n = 2, 9%). Prevalence of the idiopathic causes (n = 25) was 53%.\n\nConclusions were as follows: A positive correlation between VEGF levels and recurrent abortions were found. Serum VEGF level had no relation with gestational age. Serum VEGF levels were positively correlated with maternal age. Increased maternal age above 35 years have positive relation with serum VEGF levels.28\n\n4. A study was conducted to see the essence of various cytokines on reproductive system conducted in the department of microbiology by Batool Mutar Mahdi et al. in the Al-Kindy College of Medicine, Baghdad, Iraq, on women with reproductive failure to assess serum levels of anti-inflammatory cytokines like (IL-10 and IL-6) with pro-inflammatory cytokines like tumour necrosis factor (TNF-a) and interferon-gamma (IFN-c).\n\nMethodology: study design was cross- sectional, workup was done at a Hospital in Bagdad, Iraq from 2008 to 2010. 45 women with unexplained infertility were taken up for the research purpose. The study group consisted of women with normal ultrasonography, hysterosalpingography, husbandâs semen analysis. Other factors of infertility like tubal, ovarian, cervical, uterine, autoimmune, peritoneal and hormonal were excluded. Serum levels of cytokines (IL-10, IL-6, IFN-gamma and TNF-alpha) were measured using ELISA and comparison with control group consisting of ethnically matched 30 fertile women were done.\n\nResults were as follows: Cytokine profile showed that there was a statistical increase in IL-10 (18.09) (P = 0.002) and IFN-c (49.62) (P = 0.0001) in patients of unexplained infertility. IL-6 and TNF-alpha showed no significant difference in fertile patients. There was no correlation in the average age of infertile women group - 32.2 ± 6.1SD (range- 20-40 years) with average age of fertile group which was 30.4 ± 9.1 SD (range - 19-39 years). There was also no correlation in BMI.\n\nConclusion was that there was significant increase in IL-10 and IFN-c in women with unexplained infertility.29\n\n5. Another study was conducted to see the correlation of pro â angiogenic factors (VEGF-A and C levels) and clinical features of women having repeated pregnancy failure and compare with healthy pregnant females of same age group and characteristics features. It was a case control study conducted in Kasturba Medical College, Manipal by Bagheri et al. in the Department of Biochemistry which included 90 non- ANC women with previous history of recurrent pregnancy losses with age-matched 2 control groups as follows: 1. 70 non-ANC women with no previous history of repeated miscarriage with minimum single live child (controls) and 2. 70 ANC mothers without previous history of repeated abortions with a minimum one live child (controls). Those with undetermined recurrent abortions were included in the study in which other factors of recurrent abortions like endocrinological, anatomical, immunological and infectious factors were ruled out. Demographic and anthropometric data were collected via preset questionnaire and serum levels of VEGF-A and C were measured by ELISA kit.\n\nResult showed that levels of VEGF-A and C were significantly less in recurrent spontaneous abortions group (189.87 ± 88.1 vs 238.8 ± 99.6) when compared with healthy non pregnant first group (239.1 ± 99.7 vs 275.5 ± 133.08) and pregnant second group (301.5 ± 76.4 vs 402.5 ± 128.6). it was also observed that the clinical characteristic factors were significantly associated with concentration of VEGF A and C levels in cases and controls. The result also showed negative correlation of serum VEGF A and C levels when compared with cases and controls in age and BMI.30\n\n6. In a study conducted to Evaluate serum levels of Pro-inflammatory Cytokines in cases of Idiopathic Repeated Pregnancy Losses by Poonam Tyagi et al., Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Prince Sattam Bin Abdulaziz University, Al-Kharj, Riyadh, Saudi Arabia, in the year 2020, a total of 100 patients of age group 21-41 years were taken up for study and were divided into two group: Fifty pregnant females with history of idiopathic recurrent abortions were taken under study group and 50 healthy pregnant women were recruited under control group. Idiopathic recurrent abortion was excluded by ruling out of autoimmune, anatomic, infectious, genetic and endocrinological causes. Control group had fifty normal healthy pregnant women who attended outpatient department of obstetrics and gynaecology for a routine check-up, having <20 weeks of gestation with previous one live birth, and without any history of the treatment for miscarriage or infertility with normal ultrasonography. Blood sample were collected for estimation of cytokines as interleukin (IL)-6, TNF â alpha, IL-18, IL-12, IL-8 and interferon (IFN)-γ using ELISA kit.\n\nThere was no relation found between all the demographic characteristics except number of abortions in case of study group patients (P Ë 0.001).\n\nFSH level was statistically significant (6.14 ± 1.32) in cases of women with recurrent miscarriage group in comparison to pregnant women of control group (9.13 ± 1.10) (P Ë 0.001).\n\nThe mean with standard deviation of the level of IL-6 cytokine level was found lower (79.63 ± 1.49) in women with recurrent abortion group in comparison to pregnant control group females (93.26 ± 1.63) with P value Ë0.001. The increased level of IL-8 cytokines (72.97 ± 1.67 pg/ml) was observed in pregnant females with previous history of idiopathic recurrent abortion as compared with normal healthy pregnant controls (64.67 ± 1.36) (P Ë 0.001). The high levels of IL-12 cytokines (51.51 ± 1.97) found in patients with previous history of abortion group was identified as compared to healthy pregnant controls (18.23 ± 3.13). The result was significant statistically (P Ë 0.001). The mean ± SD level of IL-18 type of cytokine level was found significantly increased (596.18 ± 99.16 pg/ml) in patients having a history of recurrent abortions in comparison to controls (329 ± 101.18) with (P Ë 0.001). Mean ± SD level of IFN-γ was also observed elevated significantly (216.86 ± 59.86) in serum of women of idiopathic recurrent abortions as compared with pregnant women (123.12 ± 61.81) (P Ë 0.001). Increased production of TNF-α (335.13 ± 4.97) was measured in case of study group patients with previous history of recurrent abortions in comparison to normal healthy pregnant women (180.79 ± 4.12) with (P Ë 0.001).\n\nResults: Th1 activity (IL-18, IL-8, IL-12, TNF-α, and IFN-γ) were found to be more in first group of patients with recurrent abortions irrespective of pregnancy outcome as compared with Group II controls (P < 0.001) while (IL-6) which belongs to Th2 group was found decreased in Group I patients of recurrent abortion (P < 0.001) when compared with normal control Group II.31\n\n7. In a study conducted to see the correlation between vascular endothelial growth factor and 3D doppler ultrasonography with sub endometrial wave like movement in case of unexplained infertility by Eman,M.et al., in the Department of Obstetrics and Gynaecology, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt, from September 2016 to September 2019, the Relation between Vascular Endothelial Growth Factor (VEGF),sub endometrial blood flow with doppler by 3D as indicator of uterine receptivity in spontaneous as well as stimulated cycles in unexplained infertile women were evaluated during the implantation window. Total of 96 participants were recruited. They were put into three groups. Group one was: thirty-two patients with unexplained infertility with unstimulated cycle, second group was thirty-two patients of unexplained infertile women with stimulated cycle (clomiphene citrate) and third group was thirty-two participants normal fertile seeking contraception as control group. Groups (A and C), first and third group were asked to come during mid luteal phase for examination while patients in group (B), second group, were asked to come first in follicular phase and they were given clomiphene citrate drug 50 mg twice a day for 5 days starting from day 2 of cycle. Follicles were monitored till 18 mm of size and then they were given HCG and cyclogest rectal suppository for luteal support and were called on day 21-22 to see for rupture.\n\nAll participants were called on day 21-22 day of cycle for 3D transvaginal ultrasound to detect sub endometrial blood flow, sub endometrial wave like movement and on the same day venous blood sample was also taken to measure serum VEGF.\n\nResult was found as follows: There was a statistically significant decrease in below endometrial blood flow among infertile groups (A and B) than fertile group (C) with P value <0.001. Serum levels of VEGF during peri implantation period was found to be lower in group A and B infertility group when compared to group C of fertile controls. There was statistical significance between VEGF levels and Doppler blood flow study to sub endometrial region with P value 0.023. There was no relation found in all 3 groups regarding demographic features. It was found that the serum concentration VEGF levels of <200 pg/ml was able to predict good endometrial receptivity with a sensitivity of 100% and a specificity of 96.8%.\n\nConclusion out of the study was that transvaginal colour Doppler study of the sub endometrial blood flow distribution is an effective method to evaluate endometrial receptivity. The junctional zone is to be considered as a separate functional unit within the uterine cavity and plays a significant role in the processes of implantation. Serum VEGF levels were found to rise with increasing Doppler vascular penetration zones which indicates that serum VEGF levels is a good marker to determine endometrial receptivity.32\n\n8. The study comparing serum levels of VEGF in normal early pregnant women with women of recurrent pregnancy loss was conducted in the Department of Anatomy, Lady Hardinge Medical College, New Delhi, India by Dr. Renu Baliyan et al., which was a descriptive comparative study. Total 120 pregnant patients were recruited for the study, divided in two groups: a study group (60 women) and control group (60 women). The points on which they were taken for the study group were age group between 20-35 years with 3 or more consecutive recurrent pregnancy losses. Subjects had monogamous relation with no anatomical, infective, autoimmune, hormonal, or chromosomal factors which could cause recurrent pregnancy loss. The women in control group were of same age group without any history of abortions, intra uterine demise or still births and at least one live birth. After detailed history taking and general examination with systemic examinations, the blood sample was drawn from the women for VEGF levels. The correlation of serum Vascular Endothelial Growth Factor (VEGF) with recurrent miscarriage was studied.\n\nRESULTS: The variation was statistically significant with serum levels of VEGF found to be lower in recurrent abortion group as compared to normal control group. 50% of women in study group had levels below 100 pg/ml and another 48.3% had levels between 100-200 pg/ml as compared to controls who had VEGF levels between 1000-2000 pg/ml in 65% and between 2000-3000 pg/ml in another 20%.33\n\n9. Another study dates back to year 1997 which was conducted to know the levels of VEGF in the serum as well as in the follicular fluid of patients who underwent IVF by Annette Lee et al., at the Oregon Health Science University, Portland and Oregon Regional Primate Research Centre, Beaverton, Oregon, U.S.A. The study was conducted to assess the relationship of VEGF in the serum and follicular fluid (FF), Estradiol levels, and Progesterone levels in patients subjected to IVF; then to assess in early pregnancy the relationship between beta Hcg and serum VEGF levels; and also to observe and manage a case of severe ovarian hyperstimulation syndrome (OHSS) admitted for observation and management when she was subjected to IVF with her serial measurement of VEGF levels in serum and ascitic fluid. Study design was a prospective observational study. Women who underwent conventional In vitro fertilization (IVF), receiving either donor oocytes or who conceived spontaneously were included in the study. One patient had to be hospitalized with severe OHSS during the course of study period was also included to see her response and management.\n\nThis study had three parts. In first part, serum sample and Follicular fluids were obtained from patients who underwent IVF, 34 to 36 hours after ovulation during egg retrieval. In second part, women who conceived after autologous fresh embryo transfer (ET) or those who underwent IVF from the donor eggs or even women who conceived spontaneously after unstimulated cycles were taken for serum samples during the late luteal phase or early pregnancy. In third part, a patient who became pregnant and later landed up in severe OHSS following the IVF cycle done for male factor infertility was admitted, her serum and ascitic fluid samples were taken. A total of 36 patients; age between 25 to 42 years, who underwent ovarian stimulation for IVF according to a standardized pre-set protocol were studied.\n\nFor women under part 1 of study, Leuprolide or nafarelin acetate were given during the mid-luteal phase of previous cycle as pituitary down-regulation drug starting from 3 rd day of induced menses. Urinary FSH dose was individualised and given. Serial monitoring of the follicular size by serum E2 levels and transvaginal ultrasonography were done. hCG was administered 7500 IU, when atleast 2 follicles reached 18 mm diameter for follicular rupture. Oocyte collection was done 36 hours later under ultrasonographic guidance. Embryo transfers were done on day 3 after eggs retrieval. In 13 patients, peripheral venous samples were drawn before oocyte retrieval, centrifuged, and the serum stored for evaluation of E2, VEGF and P. Follicular Fluid (FF) from the largest follicle seen in the mid cycle was taken from all the cases and the supernatant were stored for further evaluation of E2, P and VEGF levels. In another 6 patients, FF were separately collected from each ovary based on single largest follicle. So, a total of 42 follicular fluid samples were obtained from 36 procedure of egg retrieval. For luteal phase support, protocols were different. Patients of first group who received autologous fresh embryos were given hCG 2500 IU for 5 days after egg retrieval and 100 mg of micronized progesterone oral dose thrice a day, starting from second day of retrieval till the day of embryo transfer and intravaginal insertion thereafter. Second group women who received donor eggs were given 100 mg of oily preparation of progesterone IM daily starting from two days before Embryo transfer. Luteal phase support was given till they either had a 12 weeks pregnancy or a negative serum hCG.\n\nIn case of study group 2, women who became pregnant after autologous fresh ET (n = 8) transfer and women who received embryos from donor oocytes (n = 7), their serum samples were collected. 2 samples of serum were taken at different intervals: the first between 11-14 days and the second between 15-17 days after Embryo transfer. Recipients of donor oocytes were given GnRH agonists for pituitary down-regulation and E2 and P replacement therapy as luteal phase support. All samples of serum were analysed depending on the number of days after Embryo transfer that is (day 11-14 or day 15-17). As measure of control, serum samples from patients who underwent IVF but who did not concieve after autologous fresh ET were taken between (days 11-14; n = 8) and also from women who conceived spontaneously without any cycle of induction (n = 8). In the second group, 2 samples, starting 5 weeks from the first day of the LMP were collected 48 hours apart. It was seen that all the women who became pregnant carried their pregnancies beyond the first trimester.\n\nIn study 3, a patient who became pregnant after embryo transfer and later had triplets landed up in severe OHSS because of ovulation induction for IVF, done in view of male factor infertility. Her serum and tapped ascites Fluid samples were collected for measurement of VEGF levels. She was given 30 ampules of urinary FSH in view of ovulation induction and her serum E2 was 2,534 pg/ml on the day of hCG administration; total of 27 eggs retrieval were done. After 3 days, four fresh embryos were transferred. She presented on 7th day after ET with nausea, pain in abdomen, vomiting and moderate abdominal distention with features suggestive of hyponatraemia, ascites and haemoconcentration few days later. She was hospitalized for severe OHSS management. In view of respiratory discomfort her therapeutic paracentesis was performed twice. Serial serum as well as ascites fluid samples were collected and evaluated for VEGF level estimation. For the purpose of comparison, peritoneal fluid sample were also collected from 4 women between 30-35 years of age who had no pelvic pathology, who had come for elective tubal ligation during laparoscopy.\n\nResult were as follows: in study group 1, Follicular fluid VEGF concentrations of an average of 356 pg/mL in 13 patients were tenfold greater which was significant (P ~ 0.01) than serum VEGF levels with mean of 50 pg/ml at the time of oocyte retrieval. Follicular fluid levels of VEGF and P were positively correlated (P < 0.01). Follicular fluid VEGF levels also had positive correlation with serum P levels (P < 0.01) and also with patientâs age (P < 0.05) but had no correlation between serum or follicular fluid E2 levels neither with the counting of oocyte retrieved. The patients in whom serum VEGF was measured after autologous ET, those patients who later got pregnant had raised (P ~ 0.05) serum VEGF levels on 11-14 days as compared to those who did not concieve or who conceived after donor eggs transfer. However, serum VEGF levels declined significantly (P ~ 0.05) in the pregnant recipients of autologous embryos between 15-17 days. There were no statistical significance in serum VEGF levels between days 11-14 or days 15-17 in pregnant recipients of donor eggs. In case of patient who was admitted for management of OHSS, serum VEGF levels varied from 168 to 292 pg/mL during controlled ovarian stimulation before hCG administration and egg retrieval, and then it increased to a maximum of 1.165 pg/mL with the onset of symptoms (10 days after egg retrieval), before decreasing to 486 pg/mL as symptoms resolved during her hospitalization. The recurrence of symptoms at 21 days after retrieval again showed elevated VEGF levels (733 pg/mL). In contrast, two ascitic fluid samples had VEGF levels of 202 and 137 pg/mL, which were not significantly different from control values (149 + 8.7 pg/mL).\n\nConclusions drawn out of the study was that patients who underwent IVF, the levels of VEGF in follicular fluid at the time of oocyte retrieval was related positively with the depth of follicular luteinization. There was also a significant role of ovary in the serum VEGF levels during early pregnancy. High levels of serum VEGF can be a predictor in the development of OHSS features.34\n\n10. In another study done in women with recurrent implantation failure, endometriosis and abortions to measure the polymorphism in +405 G/C VEGF A after ICSI-ET Cycles by V Penchev et al., at the Department of Assisted Reproduction, Medical Centre, Bulgaria, in the year 2014-15, the main background behind conducting study was that almost one in six partners who are in reproductive age worldwide experienced infertility. In such cases, various assisted reproductive techniques (ART) can be utilized to obtain a healthy pregnancy and childbirth. But a large percentage of ART attempts fail and do not give pregnancy or even abortion. The successful implantation of embryos are dependent on various factors like trophoblastic and syncytotrophoblastic proliferation, migration of embryo towards uterine cavity, adhesion and invasion into the endometrial layers. These are mediated by various growth factors and hormones produced locally. VEGF was found as an important factor which play a vital part in human vasculogenesis and embryogenesis.\n\nMethodology of the study included study of 41 patients and 23 controls who were subjected to comparison of 3 genotypes which regulated angiogenesis. Study group had 41 patients who were divided according to the subtype of study into 3 subdivisions as patients with recurrent implantation failure, endometriosis and recurrent miscarriages and the control group had 23 participants who were naturally conceived pregnant women. Peripheral blood sample was collected for DNA typing. Genomic DNA was then recruited from blood samples using standard protocols. PCR reaction forwarded by Restriction Fragment Length Polymorphism assay (PCR-RFLP) by BsmFI restrictase were performed to obtain VEGF +405 G/C allele. RTPCR genotyping with 5â exonuclease technology and Sanger capillary sequencing were used as gold standards to confirm the result. The method used for ovarian stimulation, embryo cultivation and embryo transfer along with medications given for luteal phase support were same in all the cases.\n\nResults: The C allele frequency was found to have statistical significance in patientâs group as compared to control group (53.6% vs 24%, OR = 3.68, 95% CI, P < 0.002). There was also statistical significance in the incidence of the +405 C/C genotypes in women of patient group as compared to control group (37% vs 4%, OR = 16.2, 95% CI, P < 0.02).\n\nConclusion of the study was as follows: There was positive correlation between the VEGF A +405 C/C genotype and the factor for development of endometriosis and spontaneous abortions. In the recurrent implantation failure group, the results did not show any such correlations. The receptor polymorphism gene testing can be advised to patients with indications and can be properly followed up and managed for infertility. The study group needs to be expanded and it should include more genetic polymorphism so as to get more association with recurrent pregnancy losses, implantation failures and endometriosis.35\n\n11. In yet another study conducted by Dr. Priyanka Banerjee et al., the main factors causing vascular dysfunction in idiopathic recurrent miscarriages was conducted at Kolkata, India in the Institute of Reproductive Medicine and in the Indian Institute of Technology, Kharagpur in the year 2013. Total 66 patients were recruited for the study in age less than 35 years and Body Mass Index less than 28, with previous history of 3 or more recurrent abortions within the first 12 weeks of gestation with no pathological cause. They were done with a series of investigations to rule of probable anatomical, chromosomal, autoimmune, endocrinological, genetic and infectious causes. Any women found having any abnormal laboratory or clinical parameters were excluded from study. They were compared with 50 women of same age group who were undergoing sterilisation and had no previous history of abortions or mishaps in pregnancy as control group. The patients were followed up with serial ultrasonography (USG) to measure follicle growth from day 10 onwards till ovulation. Endometrial biopsies were obtained from all patients of both the groups in between 18th and 22 nd day of their menses after confirming ovulation under GA by D & C and sample obtained were sent for histopathological evaluation and estimation of various cytokines and growth factors in endometrium.\n\nResults were as follows: No statistical significance were seen in demographic features, BMI, serum oestrogen levels, endometrial thickness and progesterone levels in patients of study group and control group. USG doppler done of the endometrial layers showed that the S/D ratio, PI, EDV were significant in women of study group as compared to control group. However, RI was found to be comparable between the two groups.\n\nLevels of various vasodilators like eNOS, ADM and NO which were measured during implantation window were found to be less in study group patients as compared to control group. IL-1β, TNF-α, IFN-γ, TGF-β1 and PGE2 were found to be higher and IL-4, IL-10, IL-2, 6, 8 and VEGF were lower in study cases as compared with control cases. PECAM-1, which is a marker of vasculogenesis and angiogenesis, showed lower immunoreactivity in patients of study group as compared to strong immunoreactivity in control group. A significant positive correlation were obtained between IL-10 and PI (r = 0.79, P < 0.001), VEGF with EDV (r = 0.61, P < 0.001), and eNOS with EDV (r = 0.74, P < 0.001). The S/D ratio also showed a significant negative correlation between VEGF (r = -0.78, P < 0.001) and eNOS (r = -0.67, P < 0.001), respectively.36\n\n12. Another study conducted titled: Aberrant cytokine production from peripheral blood mononuclear cells in recurrent pregnancy loss by M.D.Bates, S. Quenby, K. Takakuwa, P.M.Johnson and G.S.Vince in the Department of Immunology and Department of Obstetrics and Gynaecology, University of Liverpool and Department of Obstetrics and Gynaecology, Niigata University School of Medicine, Japan.\n\nBACKGROUND of the study was: Successful pregnancy was dependent on an increase in Th2-type cytokine response, while a poor pregnancy outcome was associated with lower Th2 cytokines and an increase in Th1 cytokines. This prospective study was conducted to see the correlation between recurrent pregnancy loss and the cytokine response.\n\nMETHODS: 46 women with three or more recurrent miscarriages were recruited into the study and when they missed periods or confirm pregnancy were followed up if ultrasonography had a gestational sac with either a yolk sac or a foetus with cardiac activity between 6 to 10 weeks of gestation. Patients were followed up with ultrasonography for reassurance until 12 weeks gestation and later they received routine antenatal care. Patients with antiphospholipid antibody syndrome, endocrinological causes like (oligo-menorrhoea, abnormal thyroid function tests), chromosomal causes (maternal or paternal balanced translocation) or uterine structural abnormalities (assessed by cervical weakness only) were excluded from the study. One patient was also evaluated at eight weeks of gestation when a foetal cardiac activity was detected and two weeks late demise was diagnosed. Total 25 healthy pregnant women who underwent elective termination of pregnancy at 6-10 weeks of gestation were taken as gestationally age-matched controls. These women had no previous abortions. 11 non-pregnant women were also taken for blood sample collection at random points in the menstrual cycle.\n\nRESULTS: Production of IFN-γ was found to be low in pregnant women than in non-pregnant women and even lower in women with recurrent abortions (P0.0191). IL-10 was increased in pregnant women as compared to non-pregnant women, and increased in RPL women (P0.026). IL-4 was also increased in women with RPL (P0.0001). No differences in IFN-γ, IL-10 or IL-4 levels were obtained in RPL women who aborted later with those who gave live birth. RPL women who later became pregnant had similar concentrations of TNF-α to pregnant women, RPL women who later aborted had significantly lower levels of TNF than either pregnant women (P0.02) or non-pregnant controls (P0.0004).\n\nCONCLUSIONS: The cytokine shift, which characterizes normal pregnancy, was increased rather than decreased in RPL pregnant women.37\n\nThe study is under process, patient screening is under going and the reproductive failure patients are being screened for fitting in the criteria. Kits are being procured for the purpose of study.",
"appendix": "Data availability\n\nNo data is associated with this article.\n\n\nReferences\n\nSperoff L, Glass R, Kase N: Clinical Gynaecologic Endocrinology and Infertility. 6th ed.2012; pp. 867â945.\n\nPatel A, Sharma PS, Kumar P, et al.: Sociocultural determinants of infertility stress in patients undergoing fertility treatments. J. Hum. Reprod. Sci. 2018 April; 11(2): 172â179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCousineau TM, Domar AD: Psychological impact of infertility. Best Pract. Res. Clin. Obstet. Gynaecol. 2007; 21: 293â308. Publisher Full Text\n\nRastogi R: Role of imaging in female infertility (Dr.K.M Rai Memorial Oration Award). Indian J. Radio Imaging. 2010; 20(3): 168â173. Publisher Full Text\n\nTabibzadeh S, Babaknia A: The signals and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Hum. Reprod. 1995; 1: 179â202. Publisher Full Text\n\nTe Velde EA, Exalto N, Hesseling P, et al.: First trimester development of human chorionic villous vascularization studies with CD34 immunohistochemistry. Hum. Reprod. 1997; 12(7): 1577â1581. PubMed Abstract | Publisher Full Text\n\nNardo LG: Vascular endothelial growth factor expression in the endometrium during the menstrual cycle, implantation window and early pregnancy. Curr. Opin. Obstet. Gynecol. 2005; 17(4): 419â423. PubMed Abstract | Publisher Full Text\n\nGargett CE, Rogers PA: Human endometrial angiogenesis. Reproduction. 2001; 121(2): 181â186. Publisher Full Text\n\nFerrara N, Davis-Smyth T: The biology of vascular endothelial growth factor. Endocr. Rev. 1997; 18(1): 4â25. Publisher Full Text\n\nNeufeld G, Cohen T, Gengrinovitch S, et al.: Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 1999; 13(1): 9â22. Publisher Full Text\n\nPang L, Wei Z, LiO, et al.: An increase in vascular endothelial growth factors (VEGF) and VEGF soluble receptors-1(sFlt-1) are associated with early recurrent spontaneous abortion. PLoS One. 2013; 8(9): e75759. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClauss M: Molecular biology of the VEGF and the VEGF receptor family. Semin. Thromb. Hemost. 2000; 26(5): 561â570. Publisher Full Text\n\nNorwitz ER: Defective implantation and placentation: laying the blueprint for pregnancy complications. Reprod Biomed Online. 2006; 13(4): 591â599. PubMed Abstract | Publisher Full Text\n\nCoulam CB, Clark DA, Beer AE, et al.: Current clinical options for diagnosis and treatment of recurrent spontaneous abortion. Am. J. Reprod. Immunol. 1997; 38: 57â74. PubMed Abstract | Publisher Full Text\n\nPractice Committee of the American Society for Reproductive Medicine: Definitions of infertility and recurrent pregnancy loss. Fertil. Steril. 2013; 99: 63. Publisher Full Text\n\nJaslow CR, Carney JL, Kutteh WH: Diagnostic factors identified in 1020 women with two versus three or more recurrent pregnancy losses. Fertil. Steril. 2010; 93: 1234â1243. Publisher Full Text\n\nPractice Committee of the American Society for Reproductive Medicine: Evaluation and treatment of recurrent pregnancy loss: a committee opinion. Fertil. Steril. 2012; 98: 1103â1111. Publisher Full Text\n\nDubinsky V, Poehlmann TG, Suman P, et al.: Role of regulatory and angiogenic cytokines in invasion of trophoblastic cells. Am. J. Reprod. Immunol. 2010; 63: 193â199. PubMed Abstract | Publisher Full Text\n\nCabar FR, Pereira PP, Schultz R, et al.: Vascular endothelial growth factor and β-human chorionic gonadotropin are associated with trophoblastic invasion into the tubal wall in ectopic pregnancy. Fertil. Steril. 2010; 94: 1595â1600. PubMed Abstract | Publisher Full Text\n\nSkobe M, Hawighorst T, Jackson DG, et al.: Induction of tumour lymphangiogenesis by VEGF-C promoted breast cancer metastatis. Nat. Med. 2001 Feb; 7(2): 192â198. PubMed Abstract | Publisher Full Text\n\nPrins JR, Gomez-Lopez N, Robertson SA: Interleukin-6 in pregnancy and gestational disorders. J. Reprod. Immunol. 2012; 95: 1â14. Publisher Full Text\n\nYounis A, Hawkins K, Mahini H, et al.: Serum tumour necrosis factor-alpha, interleukin-6, monocyte chemotacticprotein-1 and paraoxonase-1 profiles in women endometriosis, pcos, or unexplained infertility. J. Assist. Reprod. Genet. 2014; 31: 1445â1451. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHill JA, Polgar K, Anderson DJ: T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA. 1995; 273: 1933â1936. PubMed Abstract | Publisher Full Text\n\nRaghupathy R, Makseed M, Azizieh F, et al.: Maternal Th1 and Th2 type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions. Cell. Immunol. 1999; 196: 122â130. PubMed Abstract | Publisher Full Text\n\nNandi A, Gudi A, Shah A, et al.: An online survey of specialistsâ undergoing in vitro opinion on first line management options for unexplained subfertility. Hum. Fertil. 2015; 18: 48â53. PubMed Abstract | Publisher Full Text\n\nWu HM, Chiang CH, Huang HY, et al.: Detection of the sub endometrial vascularization flow index by three-dimensional ultrasound may be useful for predicting the pregnancy rate for patients undergoing in vitro fertilization-embryo transfer. Fertil. Steril. 2003; 79: 507â511. PubMed Abstract | Publisher Full Text\n\nNg EHY, Chan CCW, Tang OS, et al.: Comparison of endometrial and subendometrial blood flow measured by three-dimensional power Doppler ultrasound between stimulated and natural cycles in the same patients. Hum. Reprod. 2004; 19: 2385â2390. PubMed Abstract | Publisher Full Text\n\nCohen T, Nahari D, et al.: Interleukin 6 Induces the Expression of Vascular Endothelial Growth Factor (*). Journal of Biological Chemistry, Cell Biology And Metabolism. January, 1996; 271(2): 736â741.Publisher Full Text\n\nYan SF, Tritto I, Pinsky D, et al.: Induction of Interleukin 6 (IL-6) by Hypoxia in Vascular Cells. J. Biol. Chem. 1995; 270: 11463â11471. Publisher Full Text\n\nBagheri A, et al.: Association of angiogenic cytokines (VEGF-A and VEGF-C) and clinical characteristic in women with unexplained recurrent miscarriage, International Journal of Reproduction, Contraception, Obstetrics and Gynaecology. Int. J. Reprod. Contracept. Obstet. Gynecol. 2013 Dec; 2(4): 497â502. Publisher Full Text Reference Source\n\nAl Jameil N, et al.: Evaluation of Pro-inflammatory Cytokine Level in Cases of Idiopathic Recurrent Spontaneous Miscarriage in Saudi Arabia. Biomed. Res. 2018; 29(18): 3512â3517.\n\nEman MA: Relation between vascular endothelial growth factor and 3D doppler ultrasonography with subendometrial wave like movement in case of unexplained infertility. J. Pharm. Sci. September, 2019; 60.\n\nJeph RB, Agarwal S, Jeph V, et al.: Serum vascular endothelial growth factor levels in normal early pregnancies and recurrent abortion patients. J. Evolution Med. Dent. Sci. 2018; 7(46): 4994â4996. Publisher Full Text\n\nLee A, Lane K, Richard L, et al.: Vascular endothelial growth factor levels in serum and follicular fluid of patients undergoing in vitro fertilization, American Society for Reproductive Medicine. Elsevier Science Inc; August 1997; Vol 2(No. 2).\n\nPenchev V, et al.: The Role of +405 G/C VEGF A Polymorphism in Women with Recurrent Implantation Failure, Endometriosis and Miscarriages after ICSI-ET Cycles. EC Gynaecology. 2015; 1(3): 105â111.\n\nBanerjee P, Ghosh S, Dutta M, et al.: Identification of Key Contributory Factors Responsible for Vascular Dysfunction in Idiopathic Recurrent Spontaneous Miscarriage. PLoS One. 2013; 8(11): e80940. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBates MD, et al.: Aberrant cytokine production by peripheral blood mononuclear cells in recurrent pregnancy loss. Hum. Reprod. 2002; 17(9): 2439â2444. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "190238",
"date": "20 Sep 2023",
"name": "Ferdinando Antonio Gulino",
"expertise": [
"Reviewer Expertise Obstetrics",
"infertility"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript \"Evaluation of serum VEGF-A and interleukin 6 as predictors of angiogenesis during peri-implantation period assessed by Transvaginal Doppler Ultrasonography amongst women of prior reproductive failure: a cross-sectional analytical study\" is an interesting study protocol on serum Vascular Endothelial Growth factorâA and interleukin-6 on day 21 of the menstrual cycle of women affected by infertility. The study is ambitious but the number of the study samples is too low, 21 fertile women are not enough to have a statistical significance. It could be useful to increase the number of recruited patients. Please change the term of abortions with miscarriages. The authors have to better explain which are the ultrasonographic features to define endometrial receptivity, now this explanation is too generic.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly",
"responses": [
{
"c_id": "10326",
"date": "06 Oct 2023",
"name": "priya Pratapan Nair",
"role": "Author Response",
"response": "Respected reviewer, I thank you for taking out time to review the protocol. The research study which is being conducted is in accordance with the statistics taken where the study group and control group are calculated as per incidence of infertility in Central India. The point will be noted and abortions will be rewritten as miscarriage in the new version. the doppler ultrasonography will be done in accordance with the Applebaum criteria as mentioned in the protocol. Thanks and regards. Dr. Priya Nair"
}
]
}
] | 1
|
https://f1000research.com/articles/12-284
|
https://f1000research.com/articles/12-283/v1
|
14 Mar 23
|
{
"type": "Research Article",
"title": "Knowledge and Practice of Herbal Medicine on Oral Health among Dental Personnel in Malaysia",
"authors": [
"Abedelmalek KalefhTabnjh",
"Wan Muhamad Amir W Ahmad",
"Ruhaya Hasan",
"Mohd Zulkarnain bin Sinor",
"Mauro Henrique Nogueira Guimarães de Abreu",
"Ling Shing Wong",
"Sinouvassane Djearamane",
"Siddharthan Selvaraj",
"Abedelmalek KalefhTabnjh",
"Mohd Zulkarnain bin Sinor",
"Mauro Henrique Nogueira Guimarães de Abreu",
"Siddharthan Selvaraj"
],
"abstract": "Objective: To investigate the association between knowledge and practice of medical herbs related to oral heath among staffs at School of Dental Sciences, Universiti Sains Malaysia, Malaysia. Methods: A cross-sectional study was conducted at the School of Dental Sciences, USM, on 100 lecturers, dental surgery assistants, nurses, administrative workers, and dental technicians. A set of knowledge and practice questionnaire was used in this study. Descriptive statistics, chi-square and correlation were used to analyze the data. Results: The results showed that the dental staff's knowledge of oral herbal medicines was 32.8%. This indicates that they had a moderate level of knowledge. Almost three-quarters of the employees (73%) practiced at a low level (0-25%), while only 13.85% practiced at a moderate level. The findings revealed that there was no significant relationship between knowledge and practice scores and age or gender. The results, from the other hand, revealed a significant association between practice scores and the type of occupations among staff (P=0.04). The Correlation analyses shows a moderately positive relationship between knowledge and practice. Conclusions: Dental staff had a moderate level of knowledge and a poor level of practice with regard to herbal medicines for oral health. There was a significant association between age and knowledge and there was a significant association between the knowledge and practice among the dental personnel",
"keywords": [
"Alternative medicine",
"Herbal medicine",
"Knowledge",
"Practice",
"Oral health"
],
"content": "Introduction\n\nAccording to the World Health Organization (WHO) in 2000, traditional medicine is âa sum total of the knowledge, skills and practices based on theories, beliefs and experience indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as prevention, diagnosis, improvement or treatment of physical and mental illnessâ. For thousands of years, humans have used herbs and herbal products as an important part of traditional and alternative medicine.1\n\nHerbal medicine has developed into an intriguing field of study for scholars world-wide during the last few decades.1 Over 30% of plant species are utilized in medical treatments due to their compounds, which have beneficial long-term impacts on human health, particularly in the treatment of diseases.2 According to WHO (2000), traditional medicine is used by half of the population in developed countries and by about 80% of the people in developing countries. In addition, over 25% of medicines are based on herbs and herbs.1,3\n\nProblems in the oral cavity can have a significant impact on personsâ general well-being by causing significant pain and suffering, lowering their quality of life.4,5 Caries and periodontal diseases are the most common mouth issues worldwide as a result of this issue.6 Caries was found in 85 percent of children, 75 percent of adolescents, and 98 percent of adults worldwide. In the same setting, the prevalence of severe periodontal disease, which results in tooth loss, is 15-20% in the same age group.7\n\nKim and Lean in the year 2013, conducted a study at the Hospital Universiti Sains Malaysia in Kelantan, Malaysia, to investigate the level of knowledge and practice of 460 pregnant women regarding herbal medications. It was observed that the majority of women (89.8%) had limited knowledge of herbs. Only 8.5% of women stated that they were aware of the components in their herbal medications. However, none of them were able to identify the active compounds in the herbal medications they were taking. Only 11.5% of survey participants were aware that herbal medicine can be contaminated with bacteria, mercury, or contaminants.8\n\nOften people obtain information about herbal medicine from friends and family rather than from health care providers. This was supported by a study on the use of alternative medicines by cancer patients in Malaysia9 and another on the use of alternative medicines by community members with chronic kidney disease (CKD) in northern Tanzania. This means that they respect the opinion of family members, elders, friends, traditional healers, and herbal vendors more highly.10\n\nA study conducted in Cameroon on the herbs and plants used by traditional healers to address oral health concerns showed that the people relied on 52 plants, including onion, sweet potato, and coconut. The most often prescribed medications included those for toothache, mouth sores, sore throat, mouth ulcers, abscess, bullous lesion, broken tooth, mouth thrush, dentine sensitivity, dental caries, gingivitis, tonsillitis, sinusitis, dry mouth, oral syphilis, dental extraction, and oral cancer. Common plant components such as roots, leaves, and bark are utilized to treat dental disorders.11\n\nIn Malaysia, most Malaysians agree that herbal products are fully safe, have no dangerous or harmful chemicals and have no adverse effects on other commercial pharmaceutical medications.8 The majority relied on observations, experiences, and rituals de-rived from beliefs and cultures passed down through generations.8 According to Yusof et al., 2007, more than half of Malaysians who participated in the study (56.8%) used alternative medicine to relieve orofacial pain.12\n\nThe goal of this study was to examine the level of knowledge and practice of dental staff at the USM Health Campusâs Dental School regarding medical herbs and plants relevant to oral health. Additionally, the aim was to determine whether there is a relationship between knowledge and practice of using herbs as medicine associated with oral health and sociodemographic characteristics.\n\n\nMaterials and methods\n\nThis cross-sectional study was conducted at the School of Dental Sciences, USM, in Kelantan state, Malaysia in the period between February 2018 to July 2018.\n\nThe whole dental staff at USMâs School of Dental Sciences was recognized as the studyâs reference population. Lecturers, DSAs (Dental Surgical Assistants), nurses, administrative personnel, and dental technicians were among those who responded. Only participants who are USM employees, have Malaysian nationality, and are between the ages of 18 and 60 were chosen. Anyone over the age of 60, as well as non-USM employees, were excluded from this study. All respondents who met the inclusion criteria were sampled using both probability stratified and simple random sampling methods to minimize the bias in the study. Nurses, dental technicians, administration workers, DSAs, and lecturers made up the dental personnel, split into occupational (strata) categories. The strata were then sampled using simple random sampling. Following that, subjects were chosen as respondents for this study from each stratum based on the workersâ ratio to the total number of dental staff. There were 30 nurses, 15 dental technicians, 16 administrator workers, 16 DSA, and 30 lecturers among those who participated. Sample size was calculated according to study carried out by Farooqui and team in the year 20169 and it was 91, and by adding 10% drop out the total sample size was 101. Sample size was calculated using Arifin WN. Sample size calculator (Version 2.0). The data underlying the results and the questionnaire used can be found in Figshare.28\n\nThe tool for this study was a knowledge and practice questionnaire adapted from a study done by Azriani, 2007,13 about the use of herbal medicines during pregnancy, then modified to be suitable for oral health. The modifications include eliminating the questions related to pregnant women which are not related to this study and adding questions more related to oral health. The dependent variables in this study were knowledge and practice of herbal medicines; the independent variables were sociodemographic characteristics. The questionnaire was validated through a validation process. Content validity was carried out and a group discussion with experts included a dental public health specialist, nutrition specialist, and a dentist to be sure that the content is clear, suitable and covering the topics. The comments and suggestions from the expert discussion group were reviewed to validate the content of the questionnaire. During face validation session, ten Malay people were asked to answer the questions and their feedback was taken about unclear points. After that the questionnaire was piloted among 30 subjects from the dental staff to be sure that itâs clear, easy to understand, and there are no misunderstandings. Minor corrections were done to the questionnaire by including âNo informationâ to the part (2) Question (8), and adding âIf no The questions finishedâ after Question (12) because one of the respondents wrongly continued to answer the next questions while he answered that he never used herbs for oral health.13\n\nThe self-administered questionnaire is divided into three sections: the socio-demographic profile, knowledge of herbal medicines linked to oral health, and practice of herbal medicines connected to oral health. Subjects were asked to select the appropriate answers for sex, ethnicity, education level, married status, and occupation for socio-demographic data (close-ended questions). In terms of the subjectsâ age and monthly personal income, they were asked to specify the answer (open-ended questions).\n\nThe knowledge section included five questions about herbal medicine knowledge related to oral health. The questions were about the source of information about herbs, whether or not individuals know herbs that are used for oral health, the names of herbs individuals know, how to use herbs, and the effective ingredients. Five questions were included in the practice section. These were: using herbal toothpaste, having previously used herbs, the herbs used, frequency of intake, and reason for taking herbs.\n\nFor scoring reasons, a â1â was assigned to each correct response in the knowledge domain, while a â0â was assigned to each incorrect response. A â1â was added to the herb names to indicate that the herb was identified by the subject.14 The knowledge sectionâs overall score range was â0 â 10â. In the practice domain, a â0â was assigned to did not use and was unsure, but a â1â was assigned to the yes choice. For the herbs used, a â1â was assigned to each herb that was consumed by subjects.14 For each decision made by the subject, the option âneverâ received a score of â0â, âif necessaryâ, âevery weekâ, and âevery dayâ received scores of â1â, â2â, and â3â, respectively, while âevery dayâ indicates the highest frequency.15 A â1â was assigned to each oral problem chosen by the subject in the use domain.14 Knowledge and practice scores were categorized as Poor (0â25%), Moderate (26%â50%), Good (51â75%), and Very Good (76â100%) based on the proportion of total marks received for correct answers.16\n\nWhen analyzing the data, IBM SPSS version 22.0 was used. For continuous numerical data (age and income), descriptive analysis such as mean and standard deviation was utilized, whereas frequency with percentage was used for categorical variables (sex, ethnicity, marital status, education level, and occupation). The Shapiro test was used to test the normality of the continued data. After dividing knowledge and practice scores of the participants into four groups, the Chi-square test and Fisher exact test were used to examine the associations between herbal medicine knowledge and practice groups and socio-demographic factors. A Pearson correlation test was used to explore the relationship between knowledge and practice scores.\n\nThe Human Research and Ethics Committee of Universiti Sains Malaysia granted ethical approval (USM /JEPeM/17120725). Additionally, the researcher explained the study to participants who decided to participate and got a formal consent form to ensure that they agreed voluntarily to participate. The data were kept confidential and informed written consent was received from the participants who were kept anonymous.\n\n\nResults\n\nA total of 107 respondents were invited to participate in this study; however, 100 respondents completed the study, giving a 93% response rate. The majority (98%) were Malay, while the remaining 2% were Chinese. 70% of participants were female, whereas 30% were male. The participantsâ mean (SD) age was 37.3 (7.4) years, ranging from 23 to 58 years (Table 1). There was no statistically significant difference in mean ages between males and females (p = 0.278). There were 17 participants who didnât answer the question regarding the income and 4 participants didnât answer the age question. These cases were considered as missing data while testing the association with income and age variables and were therefore excluded.\n\na Mean (SD).\n\nb Median (IQR).\n\nMore than one-third of the staff (35%) had a poor level of knowledge; another (48%) had a moderate level of knowledge, only (14%) had a good level of knowledge, and (3%) had a very good level of knowledge. The mean (SD) of knowledge score (Ks) was 3.3 (2.4), indicating that 32.8 % of dental staff were knowledgeable about herbal medicines related to oral health. This shows that they had a moderate level of knowledge of the topic. Table 2 shows the respondentsâ knowledge categories.\n\na Knowledge scores.\n\nTable 3 shows respondentsâ answers to questions about their knowledge of herbal medicines related to oral health. The internet was the primary source of information about herbal medicines for (65%). Approximately one-third (29%) of those respondents were reliant on their friends for knowledge regarding herbal medicines.\n\nWhen asked if they were familiar with any herbs used for dental health, moreover half (57%) responded yes. Miswak was once the most well-known oral health herb. 66% of respondents who said yes were aware it was used for oral health. Clove was the second most chosen herb, with 60% of respondents recognizing its oral health benefits. Mentha was named as one of the best oral health herbs by 42% of respondents.\n\nMore than half of the workers (64%) have no idea how to use herbs for oral health and only about a quarter of them were aware of the herbsâ active ingredients. Even among those who responded that they knew of the use of herbs for oral health, only 56% knew how to use them, and only 35% knew the effective ingredients.\n\nAlmost three-quarters of the staff (73%) were in poor practice (0-25%), another 21% were in a moderate level of practice (26-50%), only 6% were in a good level of practice (51-75%), and none were in a very good level of practice (76-100%). The mean (SD) of the practice score (Ps) was 2.7 (3.95), indicating that there was a low level of practice among dental personnel when it came to use of herbal medicines for oral health. Table 4 shows the respondentsâ categories of practices.\n\na Practice scores.\n\nAccording to the findings, only 25% of the staff used herbal toothpaste. Only one-third of respondents said they utilized herbs for dental health (34%). Miswak was used by the majority of responders (82.4%), whereas Mentha was used by about 52.9%. Clove was the third most popular herb, accounting for 38.2% of all uses. In terms of frequency of use, 44.1% used it only when necessary, 5.9% once per week, and 41.2% once per day. When asked why they used herbs for oral health, 70.6% said it was for oral hygiene, 38.2% for pain relief, 38.2% for teeth whitening, and 29.4% for bad breath. Table 5 shows results for the practice of using herbal medicines.\n\nThe association between knowledge and age showed a significant relationship between knowledge score and age group at the 95% confidence level. Older respondents have a higher knowledge score than younger ones, Ï2 = 9.429 (p = 0.018). There was no association between knowledge score and sex. These findings are shown in Table 6.\n\na Fisher exact test.\n\nThere is also no significant association of 95% confidence level between practice scores with age and sex. The value of fisher exact test for sex with practice score was Ï2 = 1.358 (p = 0.543), and for practice score with age it was Ï2 = 1.623 (p = 0.454). These findings are shown in Table 7.\n\na Fisher exact test.\n\nThe result of the association between knowledge scores and respondentsâ occupations showed that there was no association (p = 0.227). On the other hand, the results showed a relationship between practical scores and the respondentsâ occupations at 95% confidence (p = 0.048). Dental nurses had the highest practice level (18.3%) by occupation, followed by administrative workers (18.2 %). Table 8 shows these findings.\n\na Fisher exact test.\n\nAt a 95% confidence level, there was a significant association between knowledge and practice score groups (p < 0.001). According to the correlation test, there was a significant positive moderate degree correlation between these two variables (p = 0.001) (r = 0.449). As a result, participants with a high degree of knowledge also have a high level of practice.\n\n\nDiscussion\n\nTo our knowledge, this was the first study to examine the knowledge and practice of Malaysians in general, and dental personnel in particular about herbal medicine for oral health. According to the respondentsâ ethnic origins, 98% were Malay, and only 2% were Chinese. This may be explained by the fact that the School of Dental Sciences at USM is located in the state of Kelantan, where the majority of the population is Malay.17\n\nMost dental personnel depend heavily on the internet for information about herbal medicines (65%). This finding contrasts with studies done by Azriani13 and Kim & Lean8 on Malaysian pregnant womenâs knowledge and practice of herbal medications, which discovered that the primary source of information was their parents. While Yeong and Choong in the year 2017, conducted a study on the knowledge and features of herbal supplement use among a Malaysian population, they discovered that only 13.3% obtained information from the internet. The disparate sources could be explained by the natures of oral disorders, pregnancy, and other ailments encountered by the population. Additionally, the internet source has gained popularity in recent years due to its ease of access to all locations.18\n\nThe current study found that 24% of workers were aware of the active components in herbs, compared to 8.5%% in Kim & Leanâs study.8 The larger rate could be explained by the educational disparity between the two surveys. In Kim & Leanâs study, most women only attended secondary school, whereas respondents in this survey have a greater education degree. In terms of how to use herbs, both studies found that the majority of participants were unaware of how to do so, but if we consider only those who used herbs in this study, most of them knew how to use it. The participants had a medium level of knowledge. However, contrary to other studies, most Malaysians had a poor level of understanding of herbal treatments. The disparity in outcomes could be attributed to the participantsâ high level of education.8\n\nIn comparison to numerous previous studies evaluating physicians,19 medical practitioners,20 and other healthcare professionals such as pharmacists,21 and nurses, the findings of this study revealed that dental staff had a higher level of knowledge about herbal medicine.22,23 The disparity could be because this study solely tested for knowledge on herbal medicine related to oral health. In comparison, the other research examined participantsâ knowledge of various types of herbal medicine.\n\nThe majority of individuals did not use herbal toothpaste. Only 25% indicate that they have used it. This could be contributed to their awareness of the need for fluoridated toothpaste, as some individuals may believe that herbal toothpaste is not fluoridated. However, some herbal kinds of toothpaste on the market, such as Colgate® Herbal and Parodontax®, are fluoridated.24\n\nThis research found that dental professionals use herbal medicine for oral health at a low rate (Ps = 13.85%), which is much lower than previous studies on herbal medicine use in Malaysia.8,9,13,18 Other trials ranged from 34.3%to 51.4%, which is three to four times the practice score for this study. The disparity in practice percentages may be explained by the fact that all other research focused on the use of herbs during pregnancy, cancer treatment, or overall health. Despite this, no examination has been conducted into oral health practices in Malaysia. Additionally, the samples were diverse because no investigation on the use of herbs by dental staff was conducted. Additionally, dental personnel have easy access to dental services, eliminating the need for alternative medications such as herbs for oral health.\n\nAccording to this study, the dental staff utilized Miswak the most frequently for oral health. 87.5% of Miswak users did so for oral hygiene purposes. Tubaishat, 2005,25 discovered consistent results in a study on the usage of Miswak for oral hygiene among the Jordanian population. This popularity can be attributed to Miswakâs shown usefulness in improving dental health and to religious views in Islamic countries regarding Miswakâs oral health benefits, as the prophet Mohammad commanded Muslims to use it five times daily.26\n\nThe research showed that there was no significant relationship between dental staff knowledge and practice and sex, even though Farooqui et al. (2016) discovered a relationship between herb use and sex. The disparity in results could be attributed to the fact that Farooqui et al.9 examined herbal use for cancer treatment, not oral health. However, this studyâs findings were consistent with Tubaishat,25 who found no correlation between herb use and sex. However, there was an association between respondentsâ knowledge and age, which contradicted the research by Tubaishat and his colleagues, since they found no significant association between them.25 This contradiction may refer to the difference in the herbs used in the two types of research, as Tubaishatâs research focused exclusively on Miswak. This herb has been used for generations, whereas this research includes a wide range of herbs and some of them are not used worldwide and not as popular as Miswak. The absence of a relationship between practice and age, while the existence of one between knowledge and age, may reflect dental staffâs easy access to dental care services. In any case, Kim & Leanâs8 study discovered an association between herb use and age.\n\nThis study replicated the findings of Farooqui et al.9 in terms of the association between practice scores and occupations, with both studies indicating a significant association. However, their findings regarding the relationship between knowledge score and professions were contradictory. Farooqui et al. discovered a relationship, whereas our investigation found none. This difference could be a reference to the occupational categories, as all of the participants in this study are employed by a dentistry school. In comparison, the study by Farooqui et al. includes distinct groups such as unemployed, students, homemakers, and retired.9\n\nBy contrast, this study discovered a different association between practice scores and occupational categories. Dental nurses and administrator workers scored highest on herbal medicine practice, whereas administrator workers scored lowest on knowledge. The cause behind this is unknown, and additional research on this group may shed light on it.\n\nLeach, 2004 found that medical nurses had a high degree of practice and a positive attitude toward alternative medicines. According to which dental nurses had the highest practice score for herbal medicine, this finding may corroborate ours. At the same time, it contradicts the point that dental nurses have demonstrated a low level of practice with herbal medicines. This conflicting statement could be referring to the difference between dental and medical nurses, as well as the fact that herbal medicine is a subcategory of alternative medicine. Thus, high level of practice may be related to many forms of complementary and alternative medicine. Additionally, oral health applications are separate from those for other medical conditions.27\n\n\nConclusions\n\nThe dental staff at the School of Dental Sciences has a moderate level of knowledge and a poor level of practice in herbal medicines for oral health. There was a significant association between age and knowledge level, but not between age and practice. The relationship between knowledge and practice and sex is not statistically significant. The specific occupation of dental staff, on the other hand, affected the level of practice. There was a significant correlation between the level of knowledge and practice about herbal medicines used for oral health.\n\nFurther research is needed to determine the Malaysian populationâs knowledge and practice of herbs for oral health, as no studies on this subject have been conducted according to our understanding. Most studies focused on using herbal medicines during pregnancy, cancer treatment, and general health. Within the School of Dental Sciences at USM, group discussions and lectures on herbal medicines related to oral health may help to increase staff knowledge.",
"appendix": "Data availability statement\n\nFigshare: Knowledge and Practice of Herbal Medicine on Oral Health among Dental Personnel in Malaysia, https://doi.org/10.6084/m9.figshare.21834972.v1. 28\n\nThis project contains the following underlying data:\n\n⢠Herbal Medicine for OH Data (Spreadsheet for participants answers)\n\nThis project contains the following extended data:\n\n⢠KP regarding Herbal Medicine for Oral Health questionnaire (The questionnaire used in the study)\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication). Access to this dataset requires registration with an IEEE account, which is free.\n\n\nAcknowledgments\n\nThe researchers would like to thank the School of Dental Sciences at USM in Malaysia to conduct this study. Additionally, we would like to thank all participants from the dental school at USM for their assistance and cooperation.\n\n\nReferences\n\nKumar G, Jalaluddin M, Rout P, et al.: Emerging trends of herbal care in dentistry. J. Clin. Diagn. Res. 2013; 7(8): 1827â1829. PubMed Abstract | Publisher Full Text\n\nAlves RR, Rosa IM: Biodiversity, traditional medicine, and public health: where do they meet. J. Ethnobiol. Ethnomed. 2007; 3(1): 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWinslow LC, Kroll DJ: Herbs as medicines. Arch. Intern. Med. 1998; 158(20): 2192â2199. Publisher Full Text\n\nRanjarisoa LN, Razanamihaja N, Rafatro H: Use of plants in oral health care by the population of Mahajanga, Madagascar. J. Ethnopharmacol. 2016; 193: 179â194. PubMed Abstract | Publisher Full Text\n\nSelvaraj S, Naing N, Wan-Arfah N, et al.: Development and Validation of Oral Health Knowledge, Attitude and Behavior Questionnaire among Indian Adults. Medicina (Kaunas). 2022; 58(1). Publisher Full Text\n\nSelvaraj S, Naing NN, Wan-Arfah N, et al.: Assessment on Oral Health Knowledge, Attitude, and Behaviour and its Association with Sociodemographic and Habitual Factors of South Indian Population. Pesqui. Bras. Odontopediatria Clin. Integr. 2021; 21: e0135âe0135. Publisher Full Text\n\nJose M, Sharma BB, Shantaram M, et al.: Ethnomedicinal herbs used in oral health and hygiene in coastal Dakshina Kannada. J. Oral Health Comm. Dent. 2011; 5: 107â111.\n\nKim Sooi L, Lean Keng S: Herbal medicines: Malaysian womenâs knowledge and practice. Evid. Based Complement. Altern. Med. 2013; 2013: 1â10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarooqui M, Hassali MA, Shatar AK, et al.: Use of complementary and alternative medicines among Malaysian cancer patients: A descriptive study. J. Tradit. Complement. Med. 2016; 6(4): 321â326. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJames PB, Wardle J, Steel A, et al.: Traditional, complementary and alternative medicine use in Sub-Saharan Africa: a systematic review. BMJ Glob. Health. 2018; 3(5): e000895. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshu Agbor M, Naidoo S: Ethnomedicinal Plants Used by Traditional Healers to Treat Oral Health Problems in Cameroon. Evid. Based Complement. Altern. Med. 2015; 2015: 1â10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYusof Z, Mohamed N, Radzi Z, et al.: The problems and impacts of orofacial pain among a group of Malaysian aborigines. Ann. Dent. 2007; 14(1): 31â38. Publisher Full Text\n\nAzriani BA: The Use of Herbal Medicines During Pregnancy and Perinatal Mortality in Tumpat District, Master thesis, USM, Kelantan.2007.\n\nVaidya A, Aryal UR, Krettek A: Cardiovascular health knowledge, attitude and practice/behaviour in an urbanizing community of Nepal: a population-based cross-sectional study from Jhaukhel-Duwakot Health Demographic Surveillance Site. BMJ Open. 2013; 3(10): e002976. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoo HC, Poh BK, Ruzita AT: Development, validity, and reliability of a questionnaire on knowledge, attitude, and practice (KAP) towards whole grain among primary school children in Kuala Lumpur, Malaysia. Int. Food Res. J. 2016; 23(2).\n\nSabouhi F, Babaee S, Naji H, et al.: Knowledge, awareness, attitudes, and practice about hypertension in hypertensive patients referring to public health care centres in Khoor & Biobank. Iran. J. Nurs. Midwifery Res. 2011; 16(1): 34â40. PubMed Abstract\n\nMalaysian Department of Statistics: Population Distribution and Basic Demographic Characteristic Report 2010.2011, August, 5.Reference Source\n\nYeong SW, Choong YC: Knowledge and characteristics of herbal supplement usage among community pharmacy customers in a Malaysian population. Complement. Ther. Med. 2017; 35: 92â108. Publisher Full Text\n\nClement YN, Williams AF, Khan K, et al.: A gap between acceptance and knowledge of herbal remedies by physicians: the need for educational intervention. BMC Complement. Altern. Med. 2005; 5(1): 20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMikail CN, Heaney E, Nemesure B: Increasing physician awareness of the common uses and contraindications of herbal medicines: utility of a case -based tutorial for residents. J. Altern. Complement. Med. 9(4): 571â576. PubMed Abstract | Publisher Full Text\n\nChang ZG, Kennedy DT, Holdford DA, et al.: Pharmacistsâ Knowledge and Attitudes Toward Herbal Medicine. Ann. Pharmacother. 2000; 34: 710â715. Publisher Full Text\n\nAdib-Hajbaghery M, Hoseinian M: Knowledge, attitude and practice toward complementary and traditional medicine among Kashan health care staff, 2012. Complement. Ther. Med. 2014; 22(1): 126â132. PubMed Abstract | Publisher Full Text\n\nSand-Jecklin K, Badzek L: Nurses and nutraceuticals: Knowledge and use. J. Holist. Nurs. 2003; 21(4): 383â397. Publisher Full Text\n\nHosadurga R, Boloor VA, Rao SN, et al.: Effectiveness of two different herbal toothpaste formulations in the reduction of plaque and gingival inflammation in patients with established gingivitisâA randomized controlled trial. J. Tradit. Complement. Med. 2018; 8(1): 113â119. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTubaishat RS, Darby ML, Bauman DB, et al.: Use of Miswak versus toothbrushes: oral health beliefs and behaviours among a sample of Jordanian adults. Int. J. Dent. Hyg. 2005; 3(3): 126â136. Publisher Full Text\n\nHaque MM, Alsareii SA: A review of the therapeutic effects of using Miswak (Salvadora Persica) on oral health. Saudi Med. J. 2015; 36(5): 530â543. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeach MJ: Public, nurse, and medical practitioner attitude and practice of natural medicine. Complement. Ther. Nurs. Midwifery. 2004; 10(1): 13â21. Publisher Full Text\n\nTabnjh A:Herbal Medicine for OH data.xlsx. figshare. [Dataset].2023. Publisher Full Text"
}
|
[
{
"id": "277906",
"date": "11 Jun 2024",
"name": "Muhd Firdaus Che Musa",
"expertise": [
"Reviewer Expertise Preventive and Community Dentistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors, The research work is generally excellent. Well done!\nThe article could be enhanced with the following general comments: 1. The introduction section could benefit from additional justification for the importance of the research, particularly in light of findings from a previous study indicating that over half of the Malaysian participants resorted to alternative medicine to manage orofacial pain. 2. While it is acknowledged that there is limited evidence on this research topic, it would be beneficial to also explore the relationship between knowledge and practice by conducting a comprehensive literature review across various health fields. 3. It would be beneficial to define the inclusion criteria as follows: USM employees who have been employed for a specific duration.\nWell done. Wish you all the best. Thank you.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-283
|
https://f1000research.com/articles/11-36/v1
|
12 Jan 22
|
{
"type": "Research Article",
"title": "How academic sabbaticals are used and how they contribute to research â a small-scale study of the University of Cambridge using interviews and analysis of administrative data",
"authors": [
"Becky Ioppolo",
"Steven Wooding",
"Becky Ioppolo"
],
"abstract": "Background: Academic sabbaticals are seen as an important aspect of academic life and require considerable resources, however, little research has been done into how they are used and whether their effects can be measured. We explored these issues at the University of Cambridge.\nMethods: A mixed method approach including 24 interviews with academics, eight interviews with administrators; alongside analysis of administrative and publication data between 2010 and 2019.\nResults: Academics underline the importance of sabbaticals in providing uninterrupted time for research that is used to think, explore new ideas, master new techniques, develop new collaborations, draw together previous work, set work in a wider context, and provide personal discretion in research direction. They also highlight sabbaticalsâ contributions in allowing the beneficial effects of combining teaching and research, while mitigating some of the disadvantages. However, it is difficult to detect the effect of sabbaticals on publications using a time series approach.\nConclusions: Sabbaticals provide manifold contributions to academic research at the University of Cambridge; however, detecting and quantifying this contribution, and extending these findings requires wider and more detailed investigation.",
"keywords": [
"Sabbatical",
"research productivity",
"idea origin",
"universities",
"idea generation",
"research on research",
"teaching research nexus"
],
"content": "Introduction\n\nSabbaticals are a common feature of academic work at universities in many countries around the world, but they can be misunderstood by those outside the sector (Smith, 2020). Sabbaticals are a form of paid leave for academics in ongoing positions at established universities. By tradition, sabbaticals are earned at a rate of one term of sabbatical for every six worked. The term originated from the Greek, via Latin âSabbaticusâ: referring to the sabbath; and Leviticus 25:3-5 explains that the fields should be left fallow every seventh year: âbut in the seventh year there shall be a sabbath of complete rest for the landâ (Kee, 1993). During sabbatical leave, an academic is usually relieved of duties related to teaching and administration. The stated reason academics have traditionally received sabbatical entitlements is to advance their research. Traditionally, sabbaticals provide the time for extended periods of travel to other institutions and allow academics to immerse themselves in reading, writing, gaining inspiration.\n\nSabbaticals require significant resource: an academicâs teaching and administrative responsibilities must be taken over by other staff or backfilled through recruitment. Teaching is a key revenue-generating activity for most universities, and sabbaticals reduce the amount of teaching an institution can carry out - a particularly acute problem in times of austerity and financial constraint. This makes it all the more surprising that sabbaticals have not been systematically examined.\n\nAcademic literature investigating sabbaticals is very thin. The majority of articles we identified in our initial literature searches were reflective, editorial opinion pieces providing advice on how to approach and make the most of a sabbatical.\n\nThis paper provides an initial examination of how sabbaticals are used at one of the worldâs leading research-intensive universities. As part of a larger pilot project investigating the ways universities can use discretionary funds to support research (Ioppolo & Wooding, 2021), we investigated how sabbaticals were used and their contribution to research at the University of Cambridge. To carry out this investigation, we were provided with access to internal administrative data sources, including sabbatical dates, grant holding, and publication records.\n\n\nMethods\n\nEthical review was provided by the research ethics review board of the Department for Politics and International Studies at the University of Cambridge. Written informed consent was obtained from all interviewees by email.\n\nWe used qualitative and quantitative methods to investigate sabbatical use and their contributions at the University of Cambridge. In this initial work, we tested a variety of approaches and evolved our research strategy during the project. Our research project was supported by an advisory group (see Acknowledgements), assembled by Research England, who met with us (virtually) four times over the course of the project.\n\nWe used Dimensions â a research documents database created by Digital Science (Williams, 2018) â to identify articles containing âsabbaticalâ in the title or abstract (1,024 articles). We ranked this list by âFCRâ (field normalised citation) and examined the top 100 articles. We ranked the same list by the âRelevanceâ metric provided by Dimensions and examined the top 100 articles. Six articles appeared in both lists, so a total of 194 titles and abstracts were reviewed. For a list of these articles, see Extended data (Wooding & Ioppolo, 2021). Of these 194 articles, only nineteen contained relevant topics and were reviewed in more depth. Of these nineteen articles: three could not be sourced in full text, and eight were older than ten years old (i.e., publication date of 2009 or earlier). Therefore, the eight articles less than ten years old were reviewed in full. Of these eight articles reviewed in full, four were op-eds which were not in scope (as we were interested in empirical studies) but provided useful context for what academics see to be the contribution of sabbaticals. The remaining four articles were empirical studies of academic sabbaticals.\n\nArticles we considered to be outside the scope of our review, i.e. those that were in the list of 194 but rejected when their abstracts were reviewed, covered such areas as: (a) sabbatical use in professions that are not academic (e.g., nursing, clergy, librarians), (b) use of sabbatical as one of many âflexible workplace practicesâ, (c) academic papers for which data was collected âwhile on sabbaticalâ, or (d) first-person narratives of the authorâs recent sabbatical.\n\nWe were aware that research funding varies quite markedly between disciplines. We also wanted to ensure that whichever lens we used to capture disciplinary differences could be easily replicated by other institutions wishing to further analyse the contribution of sabbaticals. Therefore, we chose to undertake the quantitative analysis by using the Research Excellence Frameworkâs (REF) four Main Panels organising 34 Units of Assessment (UOAs), which have already been developed with the aim of capturing the diversity of disciplinary research practices while still trying to maintain as few units as possible (https://www.ref.ac.uk/panels/units-of-assessment/). Cambridge submitted to 30 of the 34 UOAs, and because this project was carried out while the REF 2021 exercise was being completed, all academics at Cambridge were assigned an UOA. Main Panels1 were also used as an organising framework for sampling academics across the University to interview and other strands of quantitative analysis.\n\nWe identified 24 academics currently employed by, or affiliated to, Cambridge to interview through a variety of approaches.\n\n1. We interviewed six academics who had won research-related prizes featured in Meho (2020). We specifically targeted recipients of academic awards in arts, humanities, social sciences, and multi-disciplinary fields because we wanted to test this as an approach to identify high quality research that did not rely on publication citation metrics. We identified email addresses for the identified academics by web searches, BI approached them by email and 100% of them agreed to be interviewed. The interview protocol used in this study can be found as Extended data (Wooding & Ioppolo, 2021).\n\n2. We interviewed eighteen academics with a range of disciplinary backgrounds and characteristics. The first two academics were identified through a preliminary analysis of an initial dataset to test the approach. Both the analysis and data were refined before the selection of the remaining sixteen academics through the fully stratified random process (as detailed below). These interviewees were selected from list of author-publication pairs in each discipline (using UOAs), for authors currently employed by Cambridge. We carried out the data analysis on the 5th April 2021 selecting all publications we had recorded in our administrative database since 1st Jan 2010 until the date of interviewee selection 1st April 2021 â this gave us 126,449 author-publication pairs.\n\nFor each author-publication pair, we extracted the following range of publication-based significance metrics (where available):\n\n⢠Dimensions Field Citation Ratio (FCR) or Scopus citation number. FCR was preferred as it is field normalised, but it has lower coverage outside the sciences. We used Scopus raw citation number in UOAs, where this increased the number of publications for which we could obtain citation figures by over 50% (we did not have access to field normalised Scopus citation figures). We used Scopus raw citation figures for UOAs B-12, C-22, D-25, D-26, D-29, D-31, D-32 and D-33.\n\n⢠Altmetric Attention Score\n\n⢠The internal quality score obtained in the process used to build the University of Cambridgeâs REF submission, which involved scoring of the publications by two or more other academics in the same UOA.\n\n⢠Whether there was a funding acknowledgement present in either the Dimensions database, or a link to a grant in University of Cambridgeâs internal databases (absent/present). For more details on the accuracy of this assessment see (Ioppolo & Wooding, 2021).\n\nWithin a given UOA, author characteristics were stratified by gender and career stage. Gender was classified into Male/Female based on Human Resource records. Career stage was broadly classified into early (REF Early Career Researcher (ECR) status), mid (by job titles âresearch fellowâ, âresearcherâ, âuniversity lecturerâ and other uncommon titles), or senior (âprofessorâ, âreaderâ and âsenior lecturerâ). The top three author-publication pairs for each combination of publication metrics and author characteristics were included in the sampling frame (on occasion less than three publications might be included if for some combinations of characteristics there were less than three papers). For example, we included the top three papers in Law by normalised citation by female, mid-career researchers that included an acknowledgement of external funding. We repeated this selecting the top three publications for all combinations of publication characteristics, author characteristics, and funding acknowledgement for all disciplines.\n\nFinally, we also included all author-publication pairs for authors on publications that were included as Underpinning Research in REF2021 impact case studies. âUnderpinning Researchâ is the research on which the impacts described in an impact case study were based, for more detail see the Guidance on Submissions for the REF2021, each case study can have more than one paper cited as underpinning research.\n\nThis whole process yielded a sampling frame of 3,701 author-publication pairs. Some authors could be included more than once if, for example, a female mid-career researcher was the author of a publication that had a high normalised citation and scored highly on the internal REF review score. This increased the chance that we would select that academic to interview.\n\nWithin each of the four Main Panels, we aimed to find four interviewees: a female author of a publication with external funding, a male author without external funding, etc. Within each Main Panelâs set of four interviewees, we aimed to have at least one early-career and one mid-career researcher. Additionally, wanted to ensure that we only sampled each UOA once (a criterion we relaxed for UOA A-01 and B-12 given their size). To achieve this, we used a modified random sampling approach to select interviewees from the sampling frame. We selected random author-publication pairs from the frame but rejected them if they did not fit with the existing set of selections. For example, once we had selected an interviewee from UOA B-10, we rejected any further selections from UOA B-10.\n\nWe identified email addresses for authors by web searches and BI then approached interviewees by email. Where interviewees declined to be interviewed, we returned to the sampling frame and carried out additional selections. In comparison to the âprizeâ interviewees described above, in this second wave of approaches, we approached 38 interviewees and had 18 acceptances; a success rate of 47%. Table 1 shows the final composition of these 18 interviewees by gender, seniority, funding acknowledgement and discipline.\n\nInterviews were semi-structured and conducted via Zoom. They typically lasted 60 minutes as we spoke about a range of issues beyond sabbaticals. Conversations were recorded and computer transcribed with Descript. Transcripts were uploaded to ATLAS.ti v9 and coded for key themes by BI, using an evolving set of themes initially derived from background reading.\n\nWe also consulted University administrators, including staff from the Research Office, HR personnel, and School Finance Managers. These interviews were largely unstructured and provided us with valuable insights into local policies and practices which are not standardised across the University, and which academics may not themselves be fully aware of.\n\nWe used administrative information from the University of Cambridge to collate the start and end dates of every sabbatical taken by University academics from 1st January 2010 to 31st December 2019. This encompassed 2,362 sabbaticals taken by 1,319 researchers, including 1,043 pairs of sabbaticals where we can determine the gap to a subsequent sabbatical. We matched the person identifiers on these sabbaticals with administrative publication records to identify all publications by sabbatical takers over the same time period at total of 61,113 publications. These records included information on the dates and types of publication for each publication. Numerical analysis was carried out using R v3.6 (R Core Team, 2021).\n\nWe examined the five most common publication types (representing 97% of publications recorded): articles, books, book chapters, working papers and conference presentations. As these publication records underpinned constructing Cambridgeâs March 2021 REF2021 submission, these records were likely to be largely complete for the publications considered most important in each discipline (for example, conference presentations are likely to be most complete in engineering and computer science).\n\nThis analysis of publication rate was complicated by two factors. First, we needed to normalise for how frequently researchers take sabbaticals and hence how often they spend in each period after sabbatical. Second, we needed to correct for the fact that for publications where the publication date was only specified as a year, our data source recorded them as being published on the 1st January of that year, over stating the number of January publications.\n\nSabbatical frequency\n\nConsidering the sabbatical frequency issue, Figure 1 shows the gap in months between researchersâ sabbaticals by Main Panel. This analysis shows there are researchers who take sabbaticals sooner than two years (six terms) after their previous sabbatical, so we cannot limit our analysis to the first two years after a sabbatical, assuming no more sabbatical time will be taken.\n\nThis means we have to account for how often researchers are in each period after a sabbatical. For example, in the extreme case, if every researcher took a sabbatical every year, then no researchers would ever be in their second year after their sabbatical. We would therefore expect to see no publications in the second year after sabbaticals; not because researchers were unproductive but because there were no researchers in this situation to publish papers. In reality, as shown in Figure 1, researchers take sabbaticals at varying intervals. The net result is that there are more instances of researchers in the first year after their sabbatical than there are researchers in the second year after their sabbatical. Figure 2 shows the number of times researchers are in each period after their sabbaticals in the dataset.\n\nThe graph shows the number of times each researcher has spent in each month post-sabbatical start date using data from sabbaticals taken between 2010 and 2019.\n\nPublication date accuracy â âthe January problemâ\n\nConsidering the second issue, the data we had available on publication dates appeared to overstate the rate of publication in the January of each year, because all publications without a recorded month are recorded as being published in January. Figure 3 shows the number of publications recorded in each month of the year.\n\nThis January excess is particularly problematic, because, as shown in Figure 4, most sabbaticals begin in October. These two factors produce a false peak of publications at a delay of four months after sabbaticals start and every twelve months thereafter. We explored various ways to improve the recording of publication dates but were unable to find solution, so as a workaround, we removed all publications recorded with a date of 1st January. This caused a slight under-representation the publication rate at four- and sixteen-months post-sabbatical.\n\n1 = January, 4 = April, 10 = October.\n\n\nResults\n\nWe found little academic literature that aims to understand how academics use sabbaticals or that aims to understand the contribution of sabbaticals. Most academic publications that mention sabbaticals are personal reflections on the authorâs experience of a single sabbatical or opinion pieces suggesting strategies for planning a successful sabbatical.\n\nSearching the entire Dimensions database, and pre-screening using citation; relevance and abstract and title review we identified only 19 relevant papers. We focussed on the nine of those published in the last ten years, for one of which we could not obtain the full text. This left us with eight papers to review: three empirical studies, one historical description of the development of sabbaticals and four in which researchers described their personal reflections or opinions of sabbaticals.\n\nWe only identified three publications from the last ten years which were empirical studies of sabbaticals, and all of these focussed on specific aspects or styles of sabbatical. A fourth paper was a historical account of the development of sabbaticals. Of the three empirical papers, Pillinger et al. (2019) used a survey and interviews to understand the best practices for âmini-sabbaticals,â which they classified as periods from two days to six months, that researchers spent away from their home unit; typically, though not exclusively, away from the researcherâs home institution. The authors conclude that âmini-sabbaticalsâ can fill a gap in training capacities in the clinical science contexts they studied and may provide a positive return on investment for institutions that operate in a network. The second, Altmann and Kröll (2018) looked beyond academia and surveyed employees of public and private companies in Germany, to understand how a workplace supervisorâs support can impact employeesâ intention to take sabbaticals. They identified an existing stigma against the idea of sabbaticals and counterintuitively, found that supervisors actively supportive of sabbaticals, reduce employee desire to take sabbaticals because these employees already felt that their supervisor is supportive of work-life balance more generally. The third, Carraher, Crocitto, and Sullivan (2014), reviewed the literature and created a framework to help faculty members decide whether a sabbatical would be feasible and identify the optimum timing. They reviewed five empirical studies on sabbaticals which used surveys of academics to assess the publication outputs or wellbeing outcomes associated with sabbaticals. Similarly to our literature search, Carraher et al. found that most of the literature âis comprised of first person, anecdotal accounts or suggestions for improving the sabbatical processâ (Carraher et al., 2014). They concluded that the decision to take a sabbatical is complex, and there are many factors that affect an academicâs judgement on the feasibility of a sabbatical.\n\nThe fourth study we reviewed was a historical perspective on the use of research leave for the purpose of offsite travel for academics at the University of Cambridge (Heffernan & Jöns, 2013). The piece describes how sabbaticals emerged in the UK (they first appeared at Harvard in the 1880s, while the first research travel from Cambridge was granted in 1919) to support international travel to, for example, witness an astronomical event or examine archaeological sites in classical empires. Over the following decades, research travel became more commonly used for visiting state-of-the-art laboratories and maintaining relationships between collaborators.\n\nThese papers show us that while there has been some empirical work aiming to understand the contribution of sabbaticals, none of this research has focussed on why sabbaticals are important or the mechanisms by which they contribute to research in universities.\n\nIn addition to these four empirical publications, we reviewed four publications from the last ten years in which researchers described their personal reflections on sabbaticals, and opinion pieces about how academics can make the best use of sabbaticals (Erren & Nise, 2010; Ling, 2020; Smith, 2020; Yarmohammadian et al., 2018). These papers covered some key themes of sabbaticals we heard about in our interviews at Cambridge: specifically, these authors mentioned that the professional obligations of academic life are relentless and that the âpublish or perishâ environment makes sabbaticals a necessary feature to keep publishing and maintain professional credibility. The articles also noted that while friends and family outside academia sometimes see sabbaticals as a glorified vacation, the authors of these pieces considered having the chance to ârecharge oneâs batteriesâ on sabbatical was necessary to prevent burnout.\n\nSabbaticals at the University of Cambridge are only available to University Teaching Officers (UTOs, tenured or tenure-track academics) and are not available to many other academic staff, for example postdocs, who are not on permanent, tenure or tenure-track posts but who nevertheless engage in teaching and research. Sabbaticals are accrued by UTOs at the rate of one term every seven and are generally taken in blocks of one to three terms (admin.cam.ac.uk, 2017) (see Figure 5). Our interviews with researchers and administrators confirmed that researchers have enormous freedom over how to use their sabbaticals. Although they must be approved by the Head of Department and the Faculty Board, this approval is largely an administrative process and generally not an assessment of the academicâs plans for their sabbatical.\n\nInterviewees told us that whether academics in particular departments use their sabbaticals is heavily influenced by the departmental and disciplinary culture. These differences are clear in the quantitative data (Wooding & Ioppolo, 2021). Sabbatical behaviour differs across the Main Panels; shorter sabbaticals are much more common in Main Panels C and D (the social sciences, arts and humanities), and in Main Panel A (clinical medicine) many sabbaticals are not taken (see Figure 5). Main Panel A includes many clinical academics who, we were told, often struggle to take sabbaticals because of the difficulty of organising cover for their clinical commitments.\n\nWe were told that the single most important aspect of sabbaticals was the provision of uninterrupted time, which academics use to think, to explore new areas, to master new techniques, to develop new collaborations and to draw together previous work or set it in a new context. This is in line with the idea that research ideas are developed more in a pattern of âslow hunchesâ rather than in single âlight-bulbâ moments (Johnson, 2010). For researchers whose research consumables costs are low, sabbaticals also provide the opportunity of personal discretion and academic freedom to work on areas they judge important, even in the absence of external funding.\n\nThink without interruption or agenda\n\nThe academic interviewees who had access to regular sabbaticals discussed how valuable it is to have large blocks of uninterrupted time to progress their new ideas and conduct categorically different kinds of investigation. Careful consideration of the limitations of a new idea can be challenging to do without a depth of time to allow for wandering thoughts. There may not be a plan as to where the investigation should travel. This can make it challenging to progress a new research idea with discrete one-hour blocks of time between teaching and administrative responsibilities.\n\nOne academic explained:\n\nâ[I canât normally do these activities unless Iâm on sabbatical] because I donât get uninterrupted spells of time to think. My day is just broken up into one-hour or two-hour slots of doing stuff, and itâs very targeted. The value of just uninterrupted time to read and go follow down rabbit holes and expandâŠI donât have to be strategic in how Iâm deploying my time. [Being on sabbatical] felt like it is like being a PhD student again, where research is fantastic.â\n\nAnother researcher told a similar story about the importance of intellectual wandering:\n\nâYou have time where you can just sit down and think for a while. I read a bit, then I see something I don't understand so I look it up, then I see something else that's kind of interesting, so I read that too. [Sabbatical is] having the time to do that. Just following, you know, following a path, and all of a sudden it leads to a new idea. You go on a sort of walk, more instinct-driven than anything else.â\n\nExplore new areas and master new techniques\n\nA second use for uninterrupted time was to follow up those initial ideas and develop new expertise of the necessary background in a subject to pursue further funding. One researcher told us about how they had an insight about a way to bring the concepts of intellectual property into anthropology. Their sabbatical enabled them to learn the necessary background to understand and conceptualise that link, which then allowed them and others to build new areas of knowledge. In another discipline, a researcher noted that often in the early stages of developing a new research idea, it would be impossible to get external funding because the idea is still forming. They canât even explain it to themselves at that point.\n\nThis use of sabbaticals to explore new areas also encompasses the use of sabbaticals to develop new research skills or expertise, which often provide the foundations for new projects. An example of this came from a researcher who used a sabbatical to transition from observational fieldwork to a laboratory-based experimental research portfolio. Although they had a grant to fund the set costs of the laboratory, they explained how it was crucial to have the sabbatical time alongside this to allow them to concentrate their time to set up the new laboratory.\n\nA fourth academic highlighted the importance of being able to change direction â using a literary sporting analogy â and the importance of sabbaticals for doing so:\n\nâ[The ability to change direction is really important for research] itâs often supposed that thereâs a kind of unrolling carpet: The pathway is known in advance and there are just hurdles to clear. The fact is that certainly in my field, and speaking as a historian of science, I think in most fields, research isnât like hurdles, itâs more like Alice playing croquet [where the hoops keep moving]. Without a sabbatical, you simply cannot start a new project. Because you donât have the time, at least in the humanities, to read around a relatively unfamiliar field.â\n\nGiving a personal example, this interviewee explained how sabbatical leave (combined with the âsabbatical equivalentâ teaching and administrative buyout of a fellowship) allowed them to prepare the groundwork for a project that became a five-year grant from the Arts and Humanities Research Council to catalogue and digitise the records of the Board of Longitude. This resource is now one of the most visited sites in the University of Cambridge digital library and led to a raft of publications and meetings. The academic was clear that preparatory work done during the sabbatical and fellowship was central to the success of the project:\n\nâIt was a big project, the biggest Iâd ever been involved with and very successful. [It would have been] impossible without the previous 18 monthsâ preparation because it was an area Iâd done no work on. At all. [Prior to the sabbatical] I had no track record in this area whatsoever.â\n\nDevelop new collaborations\n\nSome researchers mentioned using sabbaticals to travel abroad to do fieldwork, to allow them to work with collaborators for extended periods, and to attend conferences without teaching and administrative responsibilities requiring them to stay in Cambridge. These activities would not be possible without a period of dedicated research time like a sabbatical where distractions are limited. In this context, they also highlighted a synergy between teaching and research: having the skills to teach courses at the institution they are visiting allows academics on sabbatical to meet more people over a longer period of time (a whole term instead of a week during term breaks) and increase their chances of developing new collaborations.\n\nDraw together previous work\n\nA researcher from the humanities highlighted the importance of having time to re-immerse themselves in their wider collection of data from a range of projects:\n\nâI think [the sabbatical] gave me the [time to] go back to my data and really ask grounded questions. [Our discipline emphasises letting] the data determine your questions rather than starting with a question. The question changes because what you find is not what you could have predicted. So, I think that the ability to go back to the data and sort of ask in an honest way, or what actually is significant about this, and thatâs something that takes months, really months or years to do.â\n\nOne researcher who has not yet been eligible for sabbatical told us: âIâve got loads of unwritten up papers where some of the analysis has been presented at a conference, but I havenât had time to write in a journal article. So, when I get the sabbatical, Iâll try and write up some of those.â\n\nSet oneâs work in another context\n\nThe opportunity of focussed time can also allow researchers to frame their ideas in a wider context and present them to a wider audience. One researcher reported how two terms of sabbatical were used to write a popular science book and explained the motivation to do this:\n\nâI wanted to write a book about [my research programme] as a synthesis of quite a big literature. And then I thought thereâs no point in spending ages writing this if about 12 [experts] read it, [and] because itâs exciting and interesting to a broader audience⊠[The project] took longer than I expected because Iâve had to learn how to write for a different audience. But I found it incredibly rewarding, and it has transformed my teaching upon return - my teaching skills have [improved].â\n\nProvide personal discretion in research direction\n\nWhere research requires little in the way of consumables, sabbaticals provide the freedom to pursue ideas that arenât currently of interest to external funders. One researcher told us how the time allows for contribution to areas of research which are not favoured or are âunpopularâ for external funding but are still valuable avenues of inquiry to contribute to the discipline:\n\nâWhat [external funders] tend to want to do is policy related. And there are no non-ideological answers to that, and Iâm just not really that interested in going down those kinds of rabbit holes. What I want to do actually is much more reflective and âblue skyâ: theoretical but applied to policy. There are not huge amounts of funding interest in that.â\n\nEarly in our project, our advisory group suggested the importance of investigating the interrelations between teaching, research and sabbaticals. Our interviews highlighted the benefits and challenges of integrating teaching and research and how sabbaticals help mitigate the key challenge of providing a respite from the time fragmentation produced by teaching.\n\nAt the University of Cambridge, tenure-track university academics manage their own research portfolio in combination with a set of teaching responsibilities for undergraduates and post-graduate students. When we explored the reciprocal influences of teaching and research with our interviews, they highlighted benefits and tensions. This reflects an ongoing debate in the literature about whether performance in teaching and research are positively correlated, known as the teaching-research nexus (Daumiller & Dresel, 2018; Marsh & Hattie, 2002; McKinley et al., 2020; Stappenbelt, 2013; Taylor, 2007).\n\nOur interviewees noted how their involvement in teaching, at least partly allowed by their sabbaticals away from teaching, contributes to their research in a number of ways â principally by maintaining their breadth of expertise. One of the most consistent themes shared by academics across different disciplines was the belief that their teaching commitments force them to read outside their areas of research expertise. One academic spoke about feeling obligated to keep presentations to students updated with the most recent literature year on year. This led them to spend more time reading than they might otherwise do, despite recognising its intrinsic benefit. In essence, teaching âstops you from forgetting all the things you learned as a studentâ.\n\nIn particular, being involved in teaching requires academics to have a sufficiently deep understanding of the material in the curriculum to be able to teach it. One academic told us: âTeaching has required that I covered a much wider range of theoretical issues than I might otherwise have done, [thereby] becoming interested in those open new avenues.â Another academic mentioned that the benefits of being involved in teaching include: âit helps [me] clarify the arguments, the cause and effects, the linkages and sets up clearly in [my] mind what it is that [I am] sayingâ.\n\nAcademics noted that there is a distinct difference between being able to teach in more specialised topics that align with oneâs research agenda compared to teaching first-year undergraduates. But in almost all cases, academics still reported that they enjoyed teaching at least to some extent and noted the benefit that the interaction of teaching helped them to feel more integrated within the University community.\n\nAcademics also felt their teaching is improved by being involved in research. They talked about how incorporating their research into the curriculum âexcites the students,â âfosters creativity in the students,â and that it benefits the students to see âwhere knowledge is being produced actively and how it's being producedâ.\n\nAs noted in the section above, academics felt that the key conflict between teaching and research was the time taken by teaching and the way teaching (and administrative) work takes time that could otherwise be used for research, one commented âsometimes it does feel like [teaching] takes time away from my researchâ. They particularly highlighted how teaching and administrative commitments broke up their time and took headspace away from thinking about research. Another academic noted, âI canât think because my day is broken up into little bitsâ.\n\nGiven this tension, a number of our interviewees highlighted how regular sabbaticals were key to providing the combination of teaching-based inspiration and breadth, alongside opportunities for extended periods of uninterrupted thought without teaching and administrative obligations. One academic described the degree of the necessity of sabbaticals:\n\nâI think the administrative burden on [academics] in this University has increased to such an extent that in-term research has become impossible. Where one used to be able to plan around sabbaticals, [sabbaticals are] now indispensable. Yes, without them I could not do research, and that's true for the vast majority of my colleagues⊠We're talking about [sabbaticals acting as] lifebelts rather than flight.â\n\nAnother academic provided this perspective on the importance of sabbaticals in providing relief from demanding teaching duties:\n\nâThe knowledge of having a term off is great. It puts you at ease and sometimes helps in situations [of intense teaching demands] where you think: Why am I doing this? [Knowing a sabbatical is coming gives] you that light at the end of the tunnel.â\n\nIn essence, sabbaticals allow university researchers to effectively combine teaching, which maintains their exposure to a broader range of literature, individuals and ideas, often the feedstock for new ideas, with uninterrupted time to conceive and explore those research ideas.\n\nAfter hearing from the interviewees about how they use sabbaticals, we combined the data on the timing of all University researchersâ sabbaticals and their publication record, to look for changes in the numbers of publication after sabbaticals. When we examined the raw publication rate post-sabbatical, on a yearly timescale (to avoid the January publication surplus issue), we saw an increase in publication rate following sabbaticals (Figure 6). However â this effect appears to be largely due to the larger number of times researchers spend in their first years after sabbatical, providing them more opportunities to publish at these times (as examined in the Methods section).\n\nWhen we normalised publication rate by dividing the publication rate by the number of times researchers spend in each period after sabbatical, the increases in publication post-sabbatical where no longer visible (Figure 7). Because most researchers who take sabbaticals take them relatively frequently, there are fewer researchers five or six years after their sabbaticals, so the normalised publication rates become increasingly stochastic as the time after sabbatical increases.\n\nTo check if there was a more rapid effect of sabbaticals, we examined the normalised publication rates on a monthly timescale while excluding 1st January publications. In the months after the start of a sabbatical, there is a suggestion that the rate of working paper publications peaks after sabbaticals (Figure 8). This analysis adds some weight to the idea that sabbaticals allow the generation of new ideas. There is also a suggestion of a slight increase in the rate of book and chapter publications during the months following the start of a sabbatical; however, we suspect that, if present, this does not represent new ideas, but the completion of previous work. We note, though, that this phenomenon could also be expected to apply to journal articles, however, here there might be a decreased publication rate in the first few months of sabbatical.\n\n1st January publications excluded because of data quality concerns. Trend line for normalised publication rate calculated for each month using a local polynomial regression implemented as LOESS).\n\n\nConclusions\n\nThis small-scale, mixed-methods study of the contributions of sabbaticals provide to the university environment contributes to the understudied area of sabbaticals. We found that sabbatical use varies by discipline, but the contributions that sabbaticals provide have same central themes across all disciplines.\n\nOur interviewees (and the limited literature) make clear the importance of uninterrupted time for generating new ideas and allowing the exploration of new areas and changes of direction. We were told how uninterrupted time is also valuable to research synthesis projects and building collaborations. Our interviewees described how the sabbatical system helps square the circle of effectively integrating teaching and research. Sabbaticals provide windows of uninterrupted time for research, with the time between sabbaticals providing the intellectual benefits of teaching. Our interviewees were very clear on the contributions of sabbaticals and gave us examples of how the uninterrupted time and freedom to think âaround the subjectâ was central to their ability to conceive significant and innovative research ideas and develop and nurture them. Often they could also link new books, research themes and papers to those sabbaticals.\n\nHowever, it is much harder to detect this relationship quantitatively in research metrics. We could only identify a clear indication of a productivity increase for the (relatively uncommon) publication type of working papers. We suspect the difficulty of detecting a relationship with other publication types is because of the long and variable timescales of book publishing, and because changes at the journal article level may be more about changes in subject matter rather than changes in volume of publication. The patterns of externally funded research leave may also add noise to any signal related to sabbaticals.\n\nGiven the strongly held beliefs and compelling examples of benefits provided by researchers in our qualitative work, and the significant cost of sabbaticals, we suggest it would be beneficial to develop a better understanding of the contribution of sabbaticals. Two potential areas for further investigation are:\n\n1) Mapping the range of sabbatical practice nationally and internationally as universities around the world will have different practices on sabbaticals and likely a different set of demonstrable benefits.\n\n2) Exploring a set of individual cases to test whether the quantitative impact of sabbaticals can be teased out of the noise and vagaries of academic publishing processes.\n\nA better understanding of the contribution provided by sabbaticals could valuably inform the debate about whether we have an appropriate balance of unconstrained and pre-specified/targeted research funding.\n\n\nData availability\n\nThe interview data generated and analysed during the current study cannot be sufficiently de-identified and therefore cannot be made publicly available, due to ethical considerations. In addition, the raw bibliometric analyses data that supported the interview sampling cannot be made publicly available, due to Scopusâ and University of Cambridgeâs licence agreements. The data could potentially be made available upon reasonable request, for the purpose of further research. For this, please contact the corresponding author.\n\nHowever, the following data is available.\n\nApollo: Research data supporting [How academic sabbaticals are used and how they contribute to research â a small-scale study of the University of Cambridge], https://doi.org/10.17863/CAM.77152 (Wooding & Ioppolo, 2021)\n\nThis project contains the following underlying data:\n\n⢠Prevalence of researchers post-sabbatical start, by month.csv\n\n⢠List of publications identified in the sabbatical contribution literature review.csv\n\nApollo: Research data supporting [How academic sabbaticals are used and how they contribute to research â a small-scale study of the University of Cambridge], https://doi.org/10.17863/CAM.77152 (Wooding & Ioppolo, 2021)\n\nThis project contains the following extended data:\n\n⢠Publications by type post-sabbatical start, by month.csv\n\n⢠Interview protocol (final).docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nThe authors acknowledge Research England for funding this study and in-kind support provided by the University of Cambridge. We would like to thank our Advisory Group: Dr Lewis Dean, Claire Packman, Steph Bales, Prof Stella Bruzzi, Dr Sarah Parks, Prof Nick Talbot, and Dr Molly Morgan Jones. In addition, we would like to thank Dr Ohid Yaqub for his advice on the development of the work and Prof Adrian Barnett for his extremely rapid comments on a draft of the paper.\n\nWe would also like to thank our colleagues at the University of Cambridge for participating in interviews and our colleagues Chris Anthony and Simon Virr in Human Resources who extracted the data for his research. Additionally, we are grateful to the many members of the University of Cambridge community and the Bennett Institute for Public Policy provided feedback throughout the project. We are particularly grateful for everyoneâs flexibility as the project was carried out over the course of three UK national lockdowns during the COVID-19 pandemic.\n\n\nReferences\n\nAltmann S, Kröll C: Understanding employeesâ intention to take sabbaticals: Analyzing the role of supportive supervisors. Pers. Rev. 2018; 47(4): 882â899. Publisher Full Text\n\nCarraher SM, Crocitto MM, Sullivan S: A kaleidoscope career perspective on faculty sabbaticals. Career Dev. Int. 2014; 19(3): 295â313. Publisher Full Text\n\nDaumiller M, Dresel M: Subjective Perceptions of the TeachingâResearch Nexus and Occupational Stress at Universities. Zeitschrift FÃŒr Entwicklungspsychologie Und PÀdagogische Psychologie. 2018; 50(3): 126â138. Publisher Full Text\n\nErren TC, Nise MS: How to have an effective academic sabbatical: Recommendations for what to do and what to avoid. Neuro Endocrinol. Lett. 2010; 31(6): 725â727. PubMed Abstract .\n\nHeffernan M, Jöns H: Research travel and disciplinary identities in the University of Cambridge, 1885â1955. Br. J. Hist. Sci. 2013; 46(2): 255â286. Publisher Full Text\n\nIoppolo B, Wooding S: Exploring the value of QR in supporting researcher-scale activities: Development of methods and a case study of the University of Cambridge. Bennett Institute for Public Policy, University of Cambridge; 2021. Publisher Full Text\n\nJohnson S: Where good ideas come from. 2010, July 13. Reference Source\n\nKee HC: The Cambridge annotated study Bible: New Revised Standard Version. Cambridge University Press; 1993. Reference Source\n\nLing DS: This pandemic is not an extended sabbatical. Nature. 2020. Publisher Full Text\n\nMarsh HW, Hattie J: The Relation between Research Productivity and Teaching Effectiveness: Complementary, Antagonistic, or Independent Constructs?. J. High. Educ. 2002; 73(5): 603â641. Publisher Full Text\n\nMcKinley J, McIntosh S, Milligan L, et al.: Eyes on the enterprise: Problematising the concept of a teaching-research nexus in UK higher education. High. Educ. 2020: 1â19. PubMed Abstract | Publisher Full Text\n\nMeho LI: Highly prestigious international academic awards and their impact on university rankings. Quantitative Science Studies. 2020; 1(2): 1â25. Publisher Full Text\n\nPillinger MH, Lemon SC, Zand MS, et al.: Come from away: Best practices in mini-sabbaticals for the development of young investigators: a White Paper by the SEQUIN (mini-Sabbatical Evaluation and QUality ImprovemeNt) Group. J. Clin. Transl. Sci. 2019; 3(1): 37â44. PubMed Abstract | Publisher Full Text\n\nR Core Team: R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2021. Reference Source\n\nSmith DR: Is sabbatical a dirty word?. EMBO Rep. 2020; 21(7): e50886. Publisher Full Text\n\nStappenbelt B: The effectiveness of the teachingâresearch nexus in facilitating student learning. Eng. Educ. 2013; 8(1): 111â121. Publisher Full Text\n\nTaylor J: The teaching:research nexus: A model for institutional management. High. Educ. 2007; 54(6): 867â884. Publisher Full Text\n\nWilliams C: Dimensions from Digital Science. Insight. 2018; 31: 33. Publisher Full Text\n\nYarmohammadian MH, Davidson P, Yeh CH: Sabbatical as a part of the academic excellence journey: A narrative qualitative study. J. Educ. Health Promot. 2018; 7: 119. Reference Source\n\nWooding S, Ioppolo B: Research data supporting [How academic sabbaticals are used and how they contribute to research â a small-scale study of the University of Cambridge] [Dataset].2021. Publisher Full Text\n\n\nFootnotes\n\n1 Main Panel A: Medicine, health and life sciences; Main Panel B: Physical sciences, engineering and mathematics; Main Panel C: Social sciences; Main Panel D: Arts and Humanities."
}
|
[
{
"id": "119613",
"date": "13 Jan 2022",
"name": "Kathryn Oliver",
"expertise": [
"Reviewer Expertise Evidence production and use"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for the chance to review this interesting piece! Really worthwhile study. A few comments:\nYou say the literature is very thin - can you provide some examples? It would also be useful in the initial paragraphs to clearly state what the questions are which you think these papers fail to address. e.g. (guessing) They fail to evaluate the cost of sabbaticals to universities, the impact on peers, careers, research productivity, teaching participation etc.\n\nI see you excluded all the 1st person narratives about sabbaticals. Would these not help in contextualising your conclusions? (maybe not of course, but good to say clearly why not).\n\nOn the interviews: I don't fully understand why you took such a complicated sampling strategy for the 18 non-prize winners. Can you explain? I assume you wanted a process to enable you to generate a sample stratified along certain criteria? What might be the downside of your approach (I'm thinking that not everyone was selected for submission to the REF for instance).\n\nOn the sabbatical analysis - you do state that requests are largely automatically granted - is this really the case? did you speak to any unsuccessful applicants? (could be reflected on in limitations if data not available). The procedure at Cambridge seems quite different to other universities (where e.g. you are eligible to take them every 5 years, for up to 1/mo per year of service).\n\nIt would also be good to reflect on the other demands on academics' time. They have a very different effect if you have a low teaching burden (which is often the case for more senior staff). I imagine this varies across the university?\nSlightly confusing language? \" However â this effect appears to be largely due to the larger number of times researchers spend in their first (initial?) years after sabbatical, providing them more opportunities to publish at these times (as examined in the Methods section).\" Would it be more accurate to say \"at Cambridge, sabbaticals tend to be short and frequently taken, so the first year after sabbatical occurs frequently. Or something similar?\nTypo? \"When we normalised publication rate by dividing the publication rate by the number of times researchers spend in each period after sabbatical, the increases in publication post-sabbatical were no longer visible\"\nI'd welcome more reflection on the points above, as well as on:\nWhat are the patterns across panels, age, career stage, and sex? What do you think might explain these patterns (given that you collated data on this, it would be good to know).\n\nAs you say, comparison with other settings and policies.\n\nCambridge is probably unusual in having departments and colleges. How does that complicate the picture?\n\nI like the idea of working papers as a measure of productivity. What about grant applications? Or research activities like conferences, roundtables, etc?\n\nWhat about the effect on wellbeing? (given that it came up in interviews).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9435",
"date": "14 Apr 2023",
"name": "Steven Wooding",
"role": "Author Response",
"response": "We appreciate the interest and time of our reviewers â and their suggestion that the piece is a valuable contribution to a sparse field. Because both reviewers commented on similar themes, we have prepared a combined response which we have submitted to both reviewers. In our response to reviewers, we have organised their comments into themes and detailed how we have responded to each set of comments. Literature review Reviewer 1: You say the literature is very thin - can you provide some examples? It would also be useful in the initial paragraphs to clearly state what the questions are which you think these papers fail to address. e.g. (guessing) They fail to evaluate the cost of sabbaticals to universities, the impact on peers, careers, research productivity, teaching participation etc. Reviewer 2: I wonder if your literature search has been wide enough. It seems very surprising that you found so little literature about the benefits, or otherwise, of a practice which has been in place in academia for a very long time. Reviewer 1: I see you excluded all the 1st person narratives about sabbaticals. Would these not help in contextualising your conclusions? (maybe not of course, but good to say clearly why not). Both Reviewers 1 and 2 requested more explanation as to why the literature review yielded so few results which we found useful. We were similarly surprised by the absence of literature but unfortunately, we donât have particular insights into what has caused it. We were specifically looking for studies that evaluated at a systems level (institutional, national, or international practice) whether and how the structure of a sabbatical advances research in ways that are fundamentally different to standard business as usual in universities. We wonder whether other evaluations of sabbaticals in universities have not been conducted because sabbaticals are a standard benefit of working in academia, and evaluators (academics) may fear discovering an undesirable result from their evaluations: that sabbaticals are not effective at advancing research. We already had first-person narratives about how sabbaticals are used from interviews we conducted at the University of Cambridge, which was the focus of our research. The first-person narratives discovered through the literature review did not take a broad, systems-level approach to their reflections; instead, they were summaries of project-specific actions taken during a sabbatical. We also preferred to draw on a set of reflections where we had control over the sampling rather than reflections in the literature. We have edited the Introduction section to clarify this. Interview sample and sampling strategy Reviewer 1: On the interviews: I don't fully understand why you took such a complicated sampling strategy for the 18 non-prize winners. Can you explain? I assume you wanted a process to enable you to generate a sample stratified along certain criteria? What might be the downside of your approach (I'm thinking that not everyone was selected for submission to the REF for instance). Reviewer 2: I was surprised you went to such a great deal of trouble to select your interview participants. Was this complexity really necessary? What would have been different if, at the other extreme, you had you simply recruited 24 people from different faculties who had been on a recent sabbatical? It was unclear if your aim was to select people who had had publications post sabbatical, although as you went to so much trouble looking for publications, I suspect this was so. If this is so, it is not explained. Reviewers 1 and 2 both commented that the sampling strategy seemed perhaps more intricate than necessary. The previous version of this article did not sufficiently emphasise that the research presented here about sabbaticals was part of a larger study on how discretionary funds are used in universities to support research (one way of which is via sabbaticals). The data we collected for the larger project served multiple purposes, which is part of the reason why such a complex sampling strategy was used. In this wider sampling strategy, our aim was to maximise diversity of interviewees across disciplines, gender and career stages, alongside minimising the scope for any of our biases to affect the selection. We wanted to ensure that we captured any differences in the way sabbaticals were used for researchers from these different groups. We have provided more context and an explanation of these reasons. Reviewer 1 noted a concern about missing out on researchers who do not submit to the REF. In REF2021, all research active academics who were research independent were required to be submitted (in contrast to REF2014 where universities could choose not to submit some researchers). This ensured we had a comprehensive sample of researchers eligible for sabbaticals. Our selection approach focussed on researchers who were producing high quality research (measured both by publications, prizes and impact) stemmed from the wider project's desire to understand how discretionary (e.g., block grant) funding to universities supports high quality research. As Reviewer 2 notes, it is possible that we would have found different results had we selected our interviewees simply by looking for researchers who had recently had sabbaticals â we partially address this question in our quantitative analysis that looked at all researchers in the university who both published and took sabbaticals. Reviewer 1: On the sabbatical analysis - you do state that requests are largely automatically granted - is this really the case? did you speak to any unsuccessful applicants? (could be reflected on in limitations if data not available). The procedure at Cambridge seems quite different to other universities (where e.g. you are eligible to take them every 5 years, for up to 1/mo per year of service). Cambridge is probably unusual in having departments and colleges. How does that complicate the picture? We have reordered the paragraphs in the section on âAnalysis of Sabbatical Uptakeâ to make clearer that sabbatical practice differs between the departments of the university. There are departments where sabbaticals are generally not taken, however, in departments where they are taken, all of our interviewees suggested that they were approved as an administrative process. We are clear in the paper that our study focussed on Cambridge, and we have added an additional section on Limitations where we refer specifically to this and the implications. Making comparisons with other institutions was unfortunately not in the scope of our project. Cambridge certainly has more generous sabbatical arrangements than many other institutions, but we suspect that the college structure makes little difference to the practice of taking sabbaticals (although it may well have effects in other areas). Other demands on academics' time Reviewer 1: It would also be good to reflect on the other demands on academics' time. They have a very different effect if you have a low teaching burden (which is often the case for more senior staff). I imagine this varies across the university? In the study, academics generally highlighted the benefits of the uninterrupted time provided by sabbaticals, but did not, in general, distinguish between the types of activities that caused the interruptions or took up their time. We asked specifically about the teaching/research nexus which we discuss, but unfortunately donât have a good source to allow us to reflect on the other demands on academics' time (except in the general sense in which we note the time provided by sabbaticals reduced these other burdens/interruptions). Wordsmithing suggestions/corrections Reviewer 1: Slightly confusing language? \" However â this effect appears to be largely due to the larger number of times researchers spend in their first (initial?) years after sabbatical, providing them more opportunities to publish at these times (as examined in the Methods section).\" Would it be more accurate to say \"at Cambridge, sabbaticals tend to be short and frequently taken, so the first year after sabbatical occurs frequently. Or something similar? Reviewer 1: Typo? \"When we normalised publication rate by dividing the publication rate by the number of times researchers spend in each period after sabbatical, the increases in publication post-sabbatical were no longer visible\" Reviewer 1 identified instances of confusing language and typos. These have been amended. We have taken the suggestion to use the word âinitialâ and noted the implication of short sabbaticals, but not the comparative text about Cambridge sabbaticals being more frequent and shorter, as we donât have the data to back up that assertion. Patterns across different demographics Reviewer 1: I'd welcome more reflection on the points above, as well as on:What are the patterns across panels, age, career stage, and sex? What do you think might explain these patterns (given that you collated data on this, it would be good to know). It would indeed be interesting to study whether there were differences in the ways sabbaticals were used across the different demographic characteristics we sampled. Our quantitative analysis did not discover any differences between disciplines and interviewees did not discuss this directly. We have added this to the new Limitations section of our paper, which we have added based on reviewersâ suggestions. Comparisons with other settings Reviewer 1: I'd welcome more reflection on the points above, as well as on: As you say, comparison with other settings and policies. Making comparisons with other settings and policies was unfortunately outside the scope of the project that was conducted. As mentioned in our new Limitations section, we think that a study that solely focussed on sabbaticals would be well placed to make such comparisons. Other measures of productivity Reviewer 1: I'd welcome more reflection on the points above, as well as on: I like the idea of working papers as a measure of productivity. What about grant applications? Or research activities like conferences, roundtables, etc? Reviewer 1 mentioned other interesting ways to capture research productivity during sabbaticals. We did attempt an analysis of grant application data â but were unable to clean the data to a sufficient extent. We have added these suggestions to the Conclusion section about future research directions. Wellbeing contributions of sabbaticals Reviewer 1: I'd welcome more reflection on the points above, as well as on: What about the effect on wellbeing? (given that it came up in interviews). We have made the explicit link between sabbaticalsâ effect on wellbeing which was added following the second block quote in the sub-section âSummary of interviews on the teaching-research nexusâ. Insight from interviews with ROO, HR, Finance Reviewer 2: You mention you also consulted University administrators, including staff from the Research Office, HR personnel, and School Finance Managers. What did they say, and/or what contribution did they make to the project? Reviewer 2 wanted to know what University administrators said in our interviews with them. These comments are woven into the Results section of the paper already and appear in the sub-section: âAnalysis of sabbatical uptakeâ. Overall, these interviews told us more about the process of sabbatical taking â for example that it was largely administrative â but didnât shed light on the ways in which sabbaticals were used. Structural points Reviewer 2: Overall, the work is a little unclear in its presentation and would benefit from re-ordering and editing. Introduction While logic suggests there might well be a connection between sabbaticals and increased publication output, this is not mentioned in your introduction. This link should be explained and backed up with references, if they exist, or else it could be proposed as part of your hypothesis. This is particularly important as you go on to explain, in great detail, selection of interviewees on the basis of their publication outputs. Somewhat related, I suggest this sentence, for example, belongs in the introduction: âEarly in our project, our advisory group suggested the importance of investigating the interrelations between teaching, research and sabbaticals.â Literature review I suggest that this be a stand-alone section ahead of methods. The section on literature review currently in Results, which unnecessarily repeats information from the earlier section, could be brought into it. Methods and Results The Results sections, while very interesting, bears little relationship to the long explanations in Methods. I suggest the Methods should be amended to tie better to the reporting of the Results. We appreciate Reviewer 1 and Reviewer 2âs suggestions about improving the links between the sections. We have added various changes which we hope make the links between sections clearer (many detailed in our earlier comments). The biggest change is to extract the Literature review into its own section preceding the Methods."
}
]
},
{
"id": "154953",
"date": "14 Nov 2022",
"name": "Katherine Christian",
"expertise": [
"Reviewer Expertise Mixed methods research into views of academic researchers in the sciences about their workplaces"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI was very happy to review this paper as the topic is valuable, particularly if there is little previous research. It was also interesting to read your findings, thank you.\nI wonder if your literature search has been wide enough. It seems very surprising that you found so little literature about the benefits, or otherwise, of a practice which has been in place in academia for a very long time.\nI was surprised you went to such a great deal of trouble to select your interview participants. Was this complexity really necessary? What would have been different if, at the other extreme, you had you simply recruited 24 people from different faculties who had been on a recent sabbatical? It was unclear if your aim was to select people who had had publications post sabbatical, although as you went to so much trouble looking for publications, I suspect this was so. If this is so, it is not explained.\nOverall, the work is a little unclear in its presentation and would benefit from re-ordering and editing.\nIntroduction While logic suggests there might well be a connection between sabbaticals and increased publication output, this is not mentioned in your introduction. This link should be explained and backed up with references, if they exist, or else it could be proposed as part of your hypothesis. This is particularly important as you go on to explain, in great detail, selection of interviewees on the basis of their publication outputs. Somewhat related, I suggest this sentence, for example, belongs in the introduction: âEarly in our project, our advisory group suggested the importance of investigating the interrelations between teaching, research and sabbaticals.â\nLiterature review I suggest that this be a stand-alone section ahead of methods. The section on literature review currently in Results, which unnecessarily repeats information from the earlier section, could be brought into it.\nMethods and Results The Results sections, while very interesting, bears little relationship to the long explanations in Methods. I suggest the Methods should be amended to tie better to the reporting of the Results.\nYou mention you also consulted University administrators, including staff from the Research Office, HR personnel, and School Finance Managers. What did they say, and/or what contribution did they make to the project?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9436",
"date": "14 Apr 2023",
"name": "Steven Wooding",
"role": "Author Response",
"response": "We appreciate the interest and time of our reviewers â and their suggestion that the piece is a valuable contribution to a sparse field. Because both reviewers commented on similar themes, we have prepared a combined response which we have submitted to both reviewers. In our response to reviewers, we have organised their comments into themes and detailed how we have responded to each set of comments. Literature review Reviewer 1: You say the literature is very thin - can you provide some examples? It would also be useful in the initial paragraphs to clearly state what the questions are which you think these papers fail to address. e.g. (guessing) They fail to evaluate the cost of sabbaticals to universities, the impact on peers, careers, research productivity, teaching participation etc. Reviewer 2: I wonder if your literature search has been wide enough. It seems very surprising that you found so little literature about the benefits, or otherwise, of a practice which has been in place in academia for a very long time. Reviewer 1: I see you excluded all the 1st person narratives about sabbaticals. Would these not help in contextualising your conclusions? (maybe not of course, but good to say clearly why not). Both Reviewers 1 and 2 requested more explanation as to why the literature review yielded so few results which we found useful. We were similarly surprised by the absence of literature but unfortunately, we donât have particular insights into what has caused it. We were specifically looking for studies that evaluated at a systems level (institutional, national, or international practice) whether and how the structure of a sabbatical advances research in ways that are fundamentally different to standard business as usual in universities. We wonder whether other evaluations of sabbaticals in universities have not been conducted because sabbaticals are a standard benefit of working in academia, and evaluators (academics) may fear discovering an undesirable result from their evaluations: that sabbaticals are not effective at advancing research. We already had first-person narratives about how sabbaticals are used from interviews we conducted at the University of Cambridge, which was the focus of our research. The first-person narratives discovered through the literature review did not take a broad, systems-level approach to their reflections; instead, they were summaries of project-specific actions taken during a sabbatical. We also preferred to draw on a set of reflections where we had control over the sampling rather than reflections in the literature. We have edited the Introduction section to clarify this. Interview sample and sampling strategy Reviewer 1: On the interviews: I don't fully understand why you took such a complicated sampling strategy for the 18 non-prize winners. Can you explain? I assume you wanted a process to enable you to generate a sample stratified along certain criteria? What might be the downside of your approach (I'm thinking that not everyone was selected for submission to the REF for instance). Reviewer 2: I was surprised you went to such a great deal of trouble to select your interview participants. Was this complexity really necessary? What would have been different if, at the other extreme, you had you simply recruited 24 people from different faculties who had been on a recent sabbatical? It was unclear if your aim was to select people who had had publications post sabbatical, although as you went to so much trouble looking for publications, I suspect this was so. If this is so, it is not explained. Reviewers 1 and 2 both commented that the sampling strategy seemed perhaps more intricate than necessary. The previous version of this article did not sufficiently emphasise that the research presented here about sabbaticals was part of a larger study on how discretionary funds are used in universities to support research (one way of which is via sabbaticals). The data we collected for the larger project served multiple purposes, which is part of the reason why such a complex sampling strategy was used. In this wider sampling strategy, our aim was to maximise diversity of interviewees across disciplines, gender and career stages, alongside minimising the scope for any of our biases to affect the selection. We wanted to ensure that we captured any differences in the way sabbaticals were used for researchers from these different groups. We have provided more context and an explanation of these reasons. Reviewer 1 noted a concern about missing out on researchers who do not submit to the REF. In REF2021, all research active academics who were research independent were required to be submitted (in contrast to REF2014 where universities could choose not to submit some researchers). This ensured we had a comprehensive sample of researchers eligible for sabbaticals. Our selection approach focussed on researchers who were producing high quality research (measured both by publications, prizes and impact) stemmed from the wider project's desire to understand how discretionary (e.g., block grant) funding to universities supports high quality research. As Reviewer 2 notes, it is possible that we would have found different results had we selected our interviewees simply by looking for researchers who had recently had sabbaticals â we partially address this question in our quantitative analysis that looked at all researchers in the university who both published and took sabbaticals. Reviewer 1: On the sabbatical analysis - you do state that requests are largely automatically granted - is this really the case? did you speak to any unsuccessful applicants? (could be reflected on in limitations if data not available). The procedure at Cambridge seems quite different to other universities (where e.g. you are eligible to take them every 5 years, for up to 1/mo per year of service). Cambridge is probably unusual in having departments and colleges. How does that complicate the picture? We have reordered the paragraphs in the section on âAnalysis of Sabbatical Uptakeâ to make clearer that sabbatical practice differs between the departments of the university. There are departments where sabbaticals are generally not taken, however, in departments where they are taken, all of our interviewees suggested that they were approved as an administrative process. We are clear in the paper that our study focussed on Cambridge, and we have added an additional section on Limitations where we refer specifically to this and the implications. Making comparisons with other institutions was unfortunately not in the scope of our project. Cambridge certainly has more generous sabbatical arrangements than many other institutions, but we suspect that the college structure makes little difference to the practice of taking sabbaticals (although it may well have effects in other areas). Other demands on academics' time Reviewer 1: It would also be good to reflect on the other demands on academics' time. They have a very different effect if you have a low teaching burden (which is often the case for more senior staff). I imagine this varies across the university? In the study, academics generally highlighted the benefits of the uninterrupted time provided by sabbaticals, but did not, in general, distinguish between the types of activities that caused the interruptions or took up their time. We asked specifically about the teaching/research nexus which we discuss, but unfortunately donât have a good source to allow us to reflect on the other demands on academics' time (except in the general sense in which we note the time provided by sabbaticals reduced these other burdens/interruptions). Wordsmithing suggestions/corrections Reviewer 1: Slightly confusing language? \" However â this effect appears to be largely due to the larger number of times researchers spend in their first (initial?) years after sabbatical, providing them more opportunities to publish at these times (as examined in the Methods section).\" Would it be more accurate to say \"at Cambridge, sabbaticals tend to be short and frequently taken, so the first year after sabbatical occurs frequently. Or something similar? Reviewer 1: Typo? \"When we normalised publication rate by dividing the publication rate by the number of times researchers spend in each period after sabbatical, the increases in publication post-sabbatical were no longer visible\" Reviewer 1 identified instances of confusing language and typos. These have been amended. We have taken the suggestion to use the word âinitialâ and noted the implication of short sabbaticals, but not the comparative text about Cambridge sabbaticals being more frequent and shorter, as we donât have the data to back up that assertion. Patterns across different demographics Reviewer 1: I'd welcome more reflection on the points above, as well as on:What are the patterns across panels, age, career stage, and sex? What do you think might explain these patterns (given that you collated data on this, it would be good to know). It would indeed be interesting to study whether there were differences in the ways sabbaticals were used across the different demographic characteristics we sampled. Our quantitative analysis did not discover any differences between disciplines and interviewees did not discuss this directly. We have added this to the new Limitations section of our paper, which we have added based on reviewersâ suggestions. Comparisons with other settings Reviewer 1: I'd welcome more reflection on the points above, as well as on: As you say, comparison with other settings and policies. Making comparisons with other settings and policies was unfortunately outside the scope of the project that was conducted. As mentioned in our new Limitations section, we think that a study that solely focussed on sabbaticals would be well placed to make such comparisons. Other measures of productivity Reviewer 1: I'd welcome more reflection on the points above, as well as on: I like the idea of working papers as a measure of productivity. What about grant applications? Or research activities like conferences, roundtables, etc? Reviewer 1 mentioned other interesting ways to capture research productivity during sabbaticals. We did attempt an analysis of grant application data â but were unable to clean the data to a sufficient extent. We have added these suggestions to the Conclusion section about future research directions. Wellbeing contributions of sabbaticals Reviewer 1: I'd welcome more reflection on the points above, as well as on: What about the effect on wellbeing? (given that it came up in interviews). We have made the explicit link between sabbaticalsâ effect on wellbeing which was added following the second block quote in the sub-section âSummary of interviews on the teaching-research nexusâ. Insight from interviews with ROO, HR, Finance Reviewer 2: You mention you also consulted University administrators, including staff from the Research Office, HR personnel, and School Finance Managers. What did they say, and/or what contribution did they make to the project? Reviewer 2 wanted to know what University administrators said in our interviews with them. These comments are woven into the Results section of the paper already and appear in the sub-section: âAnalysis of sabbatical uptakeâ. Overall, these interviews told us more about the process of sabbatical taking â for example that it was largely administrative â but didnât shed light on the ways in which sabbaticals were used. Structural points Reviewer 2: Overall, the work is a little unclear in its presentation and would benefit from re-ordering and editing. Introduction While logic suggests there might well be a connection between sabbaticals and increased publication output, this is not mentioned in your introduction. This link should be explained and backed up with references, if they exist, or else it could be proposed as part of your hypothesis. This is particularly important as you go on to explain, in great detail, selection of interviewees on the basis of their publication outputs. Somewhat related, I suggest this sentence, for example, belongs in the introduction: âEarly in our project, our advisory group suggested the importance of investigating the interrelations between teaching, research and sabbaticals.â Literature review I suggest that this be a stand-alone section ahead of methods. The section on literature review currently in Results, which unnecessarily repeats information from the earlier section, could be brought into it. Methods and Results The Results sections, while very interesting, bears little relationship to the long explanations in Methods. I suggest the Methods should be amended to tie better to the reporting of the Results. We appreciate Reviewer 1 and Reviewer 2âs suggestions about improving the links between the sections. We have added various changes which we hope make the links between sections clearer (many detailed in our earlier comments). The biggest change is to extract the Literature review into its own section preceding the Methods."
}
]
}
] | 1
|
https://f1000research.com/articles/11-36
|
https://f1000research.com/articles/12-278/v1
|
14 Mar 23
|
{
"type": "Case Report",
"title": "Case Report: Tracheal rupture of the newborn: case report and literature review",
"authors": [
"FIlip Perde",
"Theodora Voda",
"Maria Bosa",
"Cristian George Curca",
"Theodora Voda",
"Maria Bosa",
"Cristian George Curca"
],
"abstract": "Tracheal rupture in the newborn represents a rare but severe injury with a difficult therapeutic management and causality interpretation. Here we present the case of a macrosomic female newborn who presented after a dystocic delivery with a series of complications: respiratory impairment, subcutaneous emphysema, pneumothorax, anemia, and finally a diagnosed tracheal tear, which led to a short survival despite the continuous emergency care. The same issues encountered here were also found in the literature and are represented by the delayed diagnosis, the nonspecific initial clinical signs, the lack of a commonly accepted appropriate treatment, the assessment of the mechanism of rupture, the anatomical characteristics of the injury, and the patient management within the whole clinical context.",
"keywords": [
"newborn",
"tracheal rupture",
"subcutaneous emphysema",
"shoulder dystocia",
"pneumothorax",
"pneumomediastinum"
],
"content": "Introduction\n\nTracheal rupture in the newborn represents a rare and severe complication, with a high mortality rate and a difficult diagnosis.1 This complication can be caused by emergency newborn maneuvers such as obstetrical delivery maneuvers in dystocia, endotracheal intubation (ETI), and mechanical ventilation.2,3 For a complete diagnosis, it is necessary to consider a possible preexisting congenital anomaly: tracheoesophageal fistula, tracheal stenosis, or atresia.4\n\nThe windpipe wall tear causes air leakage and tissue dissection with subsequent subcutaneous emphysema, pneumomediastinum, pneumothorax, respiratory distress, cyanosis, and dyspnea.5 These can be diagnosed clinically and with a chest radiograph, especially a lateral one, which can reveal a mispositioned endotracheal tube or an accompanying clavicle fracture. A differential diagnosis with the barotrauma following mechanical ventilation should be made.2 Computed tomography (CT) is also useful, but the most accurate diagnosis is made by bronchoscopy (fibroscopy), which can assess the location and aspect of the injury.6\n\nThe treatment is either conservatory (tracheal intubation followed by expectative healing) or surgical (suture, anastomosis, or patching of the discontinuity), both accompanied by intensive care.3,7 Whatever the treatment, the mortality is high due to the unexpected complication, the preexisting impaired clinical status, which requires intensive care, and the lack of an immediate, definitive cure. Moreover, the assessment of the severity of the diagnosis and the recommendation for fibroscopy delays the treatment, contributing to an increased risk.5,8\n\nIn addition to the few cases found in the literature, that come mostly from clinical specialties (otorhinolaryngology, pediatrics, neonatology), the current case is presented mostly from an autopsy perspective, in which we compared the postmortem with the antemortem diagnosis.9\n\n\nCase presentation\n\nHere is a full-term macrosomic (birthweight 4780 g) female newborn, vaginally delivered in the morning with shoulder dystocia after a poorly monitored pregnancy, who required manual release maneuvers through rotation and traction. Besides dystocia other risk factors were identified, such as urinary tract infection during pregnancy, green amniotic fluid during birth and single loop nuchal cord.\n\nAt birth, the APGAR score was 5 and 6 at 1 and respectively 5 minutes, and postnatally, generalized cyanosis, apnea, anterior cervical subcutaneous emphysema, and limited mobility of the right upper limb (upper right limb paralysis) were noted. In the delivery room, she received oxygen therapy on a balloon mask followed by a cephalic tent in the neonatology department and needle drainage of emphysema at the right subclavicular level. 4 hours post-birth, noting that the subcutaneous emphysema, instead of remitting, extended to the head and upper chest, the transfer to a university hospital with a neonatal intensive care unit was requested for suspected pneumothorax.\n\nThe transfer took less than 1 hour, during which the patient was cardiorespiratory stable while the subcutaneous emphysema persisted. The patient arrived at the next health facility 7 hours after birth and was admitted to the neonatal intensive care unit, where a chest radiograph confirmed emphysema and revealed bilateral pneumothorax with left lung collapse (Figure 1A and B), requiring a bilateral thoracentesis with subsequent oxygen saturation and ventricular allure amelioration.\n\nAlong with this, an ETI was performed, and despite this, emphysema was accentuated, and the patientâs condition deteriorated with bradycardia and required resuscitation.\n\nThen, at 8 hours after birth, a fibroscopy through endotracheal tube (ETT) detected a rupture of the tracheal wall, followed by an emergency tracheostomy that confirmed the trachea rupture. During tracheostomy, the patient's evolution was aggravated by cardiac arrest that required resuscitation and by important blood loss (hemoglobin decreased from 21.4 to 6.8 g/dL) that required transfusion.\n\nUnder these circumstances, the pneumothorax decreased, but despite this, the general condition remains serious, and the newborn died about 12 hours after birth.\n\nGiven the fact that the death occurred in short time after surgical procedure, a medicoâlegal autopsy was required, that showed as the main features: the emergency tracheostomy with the tracheostomy tube (Figure 3B) and the pleural drain tubes one each side; an intense head cyanosis (clinically misdiagnosed as an ecchymosis due to obstetrical trauma) with subconjunctiva petechiae (Figure 2A and B); subcutaneous emphysema of the thorax, upper limbs, neck, head including the head (Figure 3A); bilateral pneumothorax with pulmonary atelectasis and hemorrhage predominantly on the left lung (Figure 4A, B, C and D). During the dissection of the esophagus, we identified an elliptical rupture (2/0.5) cm in the posterior wall of the trachea, located less than 1 cm above the carina (Figure 5A, B, C and D â in Figure 5A and B the tracheostomy tube can be seen through the rupture); no communication between the esophagus and trachea, congenital or acquired, was found.\n\n\nDiscussions\n\nTwo clinicoâpathological patterns of the trachea rupture of the newborn can be summarized as follows6:\n\nFor premature/low birth weights infants, it is described the mechanism of tracheal perforation secondary to the ETI; the ruptures can be located anteriorly (beneath the glottis) or posteriorly (above the carina), and even bronchi can be involved; apparently, the cartilaginous rings does not offer protection, as both anterior and posterior wall can rupture.4 The acute clinical sings (subcutaneous emphysema, pneumothorax) occur immediately after the ETI, without being preexistent. This mechanism should be firstly considered if the injury is detected after a cesarean, or after more than one week after birth.1\n\nFor high weight births associated with shoulder dystocia, injuries of the brachial plexus, the trachea is mostly injured by a traction mechanism during delivery, and the localization can be anterior subglottic (more frequent) but also inferior, even circumferential; macrosomic neonates require more traction and excessive hyperextension of the neck; maybe there is a weak area; the carina ais fixedâa common injury is within 2.5 cm of the carina; another area is the cricothyroid membrane.5 An endotracheal intubation enlargement of the rupture after a previous undiagnosed rupture cannot be excluded.\n\nIn our case, there was a macrosomic newborn with shoulder distocia and possible right brachial plexus paralysis, which was in poor condition immediately after birth and presented extensive subcutaneous emphysema long before any ETI attempt; the most probable mechanism is represented by the releasing traction maneuvers which teared the posterior lower wall of the trachea during delivery, possible enlarged by later intubation attempts.\n\nNoteworthy that between the onset of subcutaneous emphysema (after birth) and the fibroscopy it passed almost 8 hours, maybe due to an apparent preservation of respiratory function. It is also to note the subcutaneous emphysema that was worsened by mechanical ventilation and the difficult emergency tracheostomy was complicated by an extensive local hemorrhage that aggravated the circulation.\n\nThe subcutaneous emphysema in newborns can also appear even outside of an identifiable airway tear, due to pneumothorax or pneumomediastinum, as a complication of mechanical ventilation or resuscitation. The pneumothorax itself can be life-threatening, especially if it occurs in an immature lung.10\n\nA few clinical considerations need to be taken into account: an intense blood stasis (cyanosis) can mistake as an ecchymosis, the clinical assessment of severity and the cause of the subcutaneous emphysema is challenging, the emergency surgical and obstetrical procedures involve inherent risks.\n\nRegarding the postmortem examination of the newborn, it is essential to establish the correct sequence of events, in order not to overrate the intubation incidents as the main cause, to differentiate the acute tracheal tear from chronic pressure perforation,4 to carefully dissect the postoperative tissues, and to check for other congenital anomalies: tracheal stenosis, trachea-esophageal fistula, immature lung.\n\n\nConclusions\n\nIn the presented case, the sequence of events suggests that the traumatic delivery rather than the intubation is more likely the cause of the tracheal rupture. It is possible that the intubation attempts with external ventilation aggravated the rupture and the abnormal passage of air into tissues with subsequent pneumomediastinum, pneumothorax, and subcutaneous emphysema.\n\nA full postmortem investigation was necessary to establish a complete diagnosis, and correctly differentiate the possible congenital anomalies from obstetrical and neonatal intensive care maneuvers.\n\nThe study was conducted according to the guidelines of the Belmont Report, Declaration of Helsinki and approved by the Ethics Committee of the National Institute of Legal Medicine Bucharest no. 4/29.12.2022. All the data was obtained from the mandatory forensic autopsy for which legal consent is not necessary. All the identifiable data regarding the patient was concealed and therefore the consent was waived by the Ethics Committee.",
"appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nDoherty KM, Tabaee A, Castillo M, et al.: Neonatal tracheal rupture complicating endotracheal intubation: a case report and indications for conservative management. Int. J. Pediatr. Otorhinolaryngol. 2005; 69(1): 111â116. Publisher Full Text\n\nAmmari AN, Jen A, Towers H, et al.: Subcutaneous emphysema and pneumomediastinum as presenting manifestations of neonatal tracheal injury. J. Perinatol. 2002; 22: 499â501. PubMed Abstract | Publisher Full Text\n\nde Lagausie P , et al.: Tracheal Rupture in a Newborn During a Complicated Delivery. Diagnosis and Surgical Repair. Eur. J. Pediatr. Surg. 1992; 2(4): 230â232. Publisher Full Text\n\nNewman B, Oh KS: Iatrogenic tracheobronchial perforation in infants. J. Thorac. Imaging. 1994; 9(4): 269â272. Reference Source\n\nKacmarynski DSF, Sidman JD, Rimell FL, et al.: Spontaneous tracheal and subglottic tears in neonates. Laryngoscope. 2002; 112(8): 1387â1393. PubMed Abstract | Publisher Full Text\n\nMahieu HF, de Bree R , Sibarani-Ponsen RD, et al.: Tracheal and laryngeal rupture in neonates: Complication of delivery or of intubation? Ann. Otol. Rhinol. Laryngol. 2004; 113(10): 786â792. Publisher Full Text\n\nBaena MF, Jurado MIF: Conservative management of tracheal rupture after intubation. Paediatr. Anaesth. 1997; 7(4): 325â327. Publisher Full Text\n\nSiegel B, Bent JP, Weinstein S: Tracheal rupture in complicated delivery: a case report and review of the literature. Int. J. Pediatr. Otorhinolaryngol. 2014; 78(10): 1784â1788. Publisher Full Text\n\nCarratola M, Hart CK: Pediatric tracheal trauma. Semin. Pediatr. Surg. 2021; 30(3): 151057. Publisher Full Text\n\nMasand M, Hauptfleisch C: Severe subcutaneous emphysema in a term neonate. Case Reports. Sep. 2018; 2018: bcr-2018-226415. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "166390",
"date": "11 Apr 2023",
"name": "Ivan Romic",
"expertise": [
"Reviewer Expertise Thoracal surgery",
"pulmology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nArticle is well written and describes a rare cause of newborn mortality which every pediatrician and gynecologist should be aware of. It explained pathophysiology and supported it with radiologic and specimen imaging.\n\nFew remarks which need to be addressed:\nYou should state exactly which maneuvers were performed to deliver the baby which could cause such serious injury\n\nI suggest to blur somehow the whole face of the baby postmortem mouth and nose) and leave just upper part of the head clear which is important for cyanosis diagnosis\n\nYou should add in discussion section the option of self expendable stents in tracheal injuries\n\nRefer to following article and add reference since it explains the connection of empyhsema, pneumothorax and pneumomediastinum in adult:Â Romic M, Becejac T, Grbavac D, Romic R, Romic I. Conservative treatment of postintubation tracheal laceration with pneumomediastinum, bilateral pneumothorax, and massive subcutaneous emphysema. Lung India. 2021 Jan-Feb;38(1):77-79.\n\nIs the background of the caseâs history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "214603",
"date": "28 Nov 2023",
"name": "Catarina Marques Duarte",
"expertise": [
"Reviewer Expertise Paediatric Intensive Care"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes a case of a tracheal lesion in a newborn, which is a rare situation and requires a high level of clinical suspicion for diagnosis. The therapeutic approach is also not consensual and there is little literature available on the approach to this pathology.\n\nIn order to complement the information described in the article, I make the following suggestions:\nGive a more detailed account of the clinical evolution and the medical approach.\nDescribe the timing of endotracheal intubation and why it wasn't carried out earlier.\n\nWas there any stability in the progression of subcutaneous emphysema after intubation?\n\nIn the delivery room, did the newborn only need oxygen therapy or was he intubated? If not, explain why.\n\nWhat type of drainage was performed? Subcutaneous emphysema drainage or pneumothorax drainage?\n\nIn order to improve the literature review component of the article, I suggest adding a paragraph on the possible therapeutic approaches to tracheal injury, surgical vs. conservative, complemented with the available literature.\n\nIs the background of the caseâs history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-278
|
https://f1000research.com/articles/12-270/v1
|
13 Mar 23
|
{
"type": "Case Report",
"title": "Case Report: Competitive causes of death: a case report of non-mutilated but fatal traumatic railway injury",
"authors": [
"Sari Nur Indahty Purnamaningsih",
"Satria Perwira",
"Prasillia Ramadhani",
"Ahmad Yudianto",
"Sari Nur Indahty Purnamaningsih",
"Prasillia Ramadhani",
"Ahmad Yudianto"
],
"abstract": "Background: Railway-related accidents can cause severe injuries, and, most of the time, death. In a country with frequent railway-related accidents, a complete autopsy is vital to determine the cause and mechanism of death, reconstruct the events before death, and identify the victim. It is also essential to determine whether the deceased died by suicide or homicide. This article reports an autopsy of a non-identified victim from a train crash with two fatal injuries. In addition, we reported the challenges in establishing the cause and manner of death. Case presentation: A man was hit by a Pertamina train on Tuesday, 19 July 2022, at 00:24 West Indonesian Time (UTC+07:00), and this was reported to the police for investigation. The body was then examined at Dr. Soetomo General Academic Hospital by a forensic expert. The external examination suggested the presence of abrasions on the face, waist, and all four limbs, lacerations on the face and right earlobe, and open head fractures. In an internal examination, skull-base fractures, fourth and sixth right rib fractures, bleeding in the chest cavity, shrinking lungs, lacerations to the liver and lower right and left lungs, and blood infiltration in the chest cavity wall muscles and scalp muscles were found. The pneumothorax test was positive on the left chest but negative for heart air embolism. Investigation of the base of the skull revealed the presence of fractures. Conclusions: Determining the cause of death in a patient with multiple injuries was challenging and required a thorough investigation. Pneumothorax and air embolism could be the primary cause of death and their presence had to be investigated. Fractures of the base of the skull were established as the cause of death. According to the investigations at the crime scene and the injury profile, suicide was concluded as the manner of death.",
"keywords": [
"autopsies",
"accident",
"pneumothorax",
"railway",
"suicidal."
],
"content": "Introduction\n\nTrains are land transportation with many benefits deriving from their efficiency in transporting passengers and goods. This mode of transportation has been utilized widely in Indonesia, connecting provinces in Sumatra, Kalimantan, Sulawesi, and mostly in Java islands. However, its safety is concerning especially with the record number of railway-related accidents. Ministry of Transportation of the Republic of Indonesia reported that, from 2015 to 2020, the number of train accidents was 130 cases.1 The train accidents that occurred over these five years caused multiple deaths and serious injuries. In 2015, an accident took the life of one person and caused three individuals to suffer serious injuries and 39 others minor injuries.1 In 2016, the total number of victims was 97, with 43 people dead, 41 people severely injured, and 13 people minorly injured.1 Furthermore, as many as 252 people were victims of train accidents in 2017, with 87 people dead, 79 people seriously injured, and 86 people lightly injured.1 Meanwhile, in 2018, there was a train accident that resulted in four victims; three were seriously injured with one dead.1 Records on train accidents found in 2019 showed 19 minorly injured individuals, and in 2020 there were no fatalities.1 However, these numbers are expected to be underreported.\n\nTraumatic injuries and disorders caused by railway accidents have medical and legal consequences, including for dead victims. The cause of death from railway-related injuries is clear in mutilated or decapitated bodies.2 However, in patients with multiple fatal injuries, establishing the cause of death is challenging. Herein, we report the case of fatal pneumothorax and base-of-skull fractures as the competitive cause of death. In addition, we report our determination on the manner of death.\n\n\nCase report\n\nOn Tuesday, 19 July 2022, at 00.24 West Indonesian Time (Coordinated Universal Time (UTC)+07:00), a Pertamina train bound for Surabaya-Malang crossed the Margorejo Shelter on Ahmad Yani Street, Surabaya, Indonesia. The train officers saw a man standing on the edge of the railway who was hit by the incoming train. After an examination and search at the crime scene, the victim was found to possess no identification documents (Figure 1a). The body was then identified at Dr. Soetomo General Academic Hospital by a forensic expert team. The external, internal, and additional investigations were conducted. The external examination suggested the presence of abrasions on the face, waist, and all four limbs (Figure 1b). Lacerations were found on the face and right earlobe. Open head fractures were also observable. The internal examination revealed the presence of lacerations on the liver, fourth and sixth right rib fractures, skull-base fractures, bleeding in the chest cavity, collapsed left lungs, and lower right and left lungs, and blood infiltration in the chest cavity wall muscles, and scalp muscles (Figure 1câe). The patientâs nail was taken for further DNA examination.\n\n(a) Photographed image of the crime scene with victim has been wrapped in a body bag before transported to Dr. Soetomo General Academic Hospital for autopsy. Identification documents were not found on the victim. External examination suggesting the presence of lacerations on the face and right earlobe and open head fractures (b). Internal examination suggesting the presence of lacerations to the liver fractures (c); on one to four left ribs, bleeding in the chest cavity, fractures on one to four left ribs, bleeding in the chest cavity, collapsed left lungs, and blood infiltration in the muscles chest cavity (d); and skull base fracture through the foramen magnum (e). Pneumothorax test on the chest and air embolism test on the heart (f-h) indicate the left chest was positive for pneumothorax but the heart was negative for air embolism.\n\nA pneumothorax test was performed on the patientâs chest. After reflecting the front portion of the chest up to the midaxillary lines, water was poured into the angle between the subcutaneous tissue flap of skin with the scalpel held in the right hand, and the intercostal tissues below the water line were pierced toward the right side of the chest. The test was negative. The same test on the left chest was conducted using a 5 mL syringe filled with water after removing the plunger. Then the needle was pierced into the pleural space and bubbles were present, confirming a positive result of the pneumothorax test. An air embolism test on the heart was also performed by opening the pericardium and filling it with water. Once the heart was fully submerged in water, the right atrium and ventricle were incised. The result turned out to be negative because of the absence of bubbles observed after the incision. The photographed images of the pneumothorax test and air embolism test are presented in Figure 1fâh, respectively.\n\nBased on the examination, the cause of death was determined to be blunt force trauma to the head, causing a fracture to the base of skull and aggravated by blunt force to the chest, resulting in suffocation.\n\n\nDiscussion\n\nThe patient herein was witnessed to be standing against the incoming train and hit by the train. The patient was found dead, with abrasions and lacerations all over the body and open head fractures. Most suicide cases have decapitation injuries because the individuals lay down on the railway to avoid the brake being pulled when they are seen by the train driver.3 However, the patient in this present case report did not suffer from decapitation, which is understandable since the patient was hit by the train while standing. The injury profile herein also had a distinct similarity with that obtained in victims who were killed crossing the rails.3â7 The suspicion toward suicide became stronger as the patient did not have an identification card and the crash occurred at night. In a previous report, it was found that most suicide cases occur at night because the victim wants to avoid being seen and saved.6\n\nAt first, we suspected a traumatic pneumothorax as the cause of death because of a positive pneumothorax test on the left chest and collapsed left lungs. Air released from the ruptured alveoli could fill up the pleural cavity, which is normally empty, resulting in pain and suffocation to the victim.2 A test on air embolism was carried out and turned out to be negative. Both a traumatic pneumothorax and air embolism could lead to death in a short amount of time.8 Further investigation at the base of the skull bones revealed the presence of fractures, located from the right front skull base, passing through the skull base hole, and to the left middle skull base bone. Therefore, the fracture of the skull base was decided as the cause of death. Skull fractures have been reported predominantly in victims who are hit by a train while walking.7 Fractures at the base of the skull have been reported as the cause of death in victims receiving a blunt force trauma.9 As a conclusion, the manner of death was determined to be unnatural in the form of suicide, and the cause of death was blunt force trauma, which resulted in a fracture of the base of skull.\n\n\nEthical approval\n\nNot required.\n\n\nConsent for publication\n\nThe authors have received written consent from the patientâs family and the Kepolisian Polrestabes Surabaya, Indonesia.",
"appendix": "Data availability\n\nAll data and materials used during this study are included in this article.\n\n\nAcknowledgements\n\nWe would like to express our sincere gratitude to all the people involved in the investigation of the case.\n\n\nReferences\n\nIndonesian MInistry of Transportation: Buku statitik bidang perkeretaapian tahun 2020. Jakarta:Kementrian Perhubungan Direktorat Jenderal Perkeretaapian;2020.\n\nTambunan E, Yudianto A, Endradita G: Death Caused by the Blunt Trauma on The Prisonerâs Chest: A Case Report. Malaysian Journal of Medicine and Health Sciences. 2021; 17: 177â179.\n\nMohanty MK, Panigrahi MK, Mohanty S, et al.: Death due to traumatic railway injury. Med. Sci. Law. 2007; 47(2): 156â160. PubMed Abstract | Publisher Full Text\n\nMartiana IK, Permana D, Widhiyanto L: Traumatic Cervical Spinal Cord Injury. is Urgent Intervention Superior to Delayed Intervention? A meta-analysis Evaluation. J. Orthop. Traumatol. Surabaya. 2019; 8(1): 12â18. Publisher Full Text\n\nSya'ban SN, Fatmaningrum W, Bayusentono S: The Profile of Fracture in Patients Under 17 Years of Age at RSUD Dr Soetomo in the Period of 2013-2014. J. Orthop. Traumatol. Surabaya. 2017; 6(1): 21â32. Publisher Full Text\n\nJibril I, Shetty BSK, Shetty P, et al.: Autopsy Death Profile Due to Railway Injury in Mangalore, A Coastal City of South Karnataka-A Retrospective Analysis. Indian J. Forensic Med. Toxicol. 2021; 15(2): 2551â2557.\n\nValsala K, Sreedevi C, Sreelekshmi J: An autopsy based comparative studyof pattern of injuries in pedestrians involved in railway track deaths. Int. J. Adv. Res. 2017; 5(1): 1070â1077.\n\nSasidharam A, Al-Kandary NM: A review of the techniques and guidelines in adult autopsies. Saudi J. Med. 2019; 04(12): 774â790. Publisher Full Text\n\nPurwanti T, Kusuma E: Head Injury Case with Blunt Force Traumatic: Case Report. New Armen. Medical J. 2022; 16(1): 109â116. Publisher Full Text"
}
|
[
{
"id": "292604",
"date": "26 Jun 2024",
"name": "Raghvendra Singh Shekhawat",
"expertise": [
"Reviewer Expertise Autopsy",
"Forensic Medicine",
"Forensic Pathology",
"Clinical Forensic Medicine",
"Forensic Toxicology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a case where fatal pneumothorax and base-of-skull fractures are the competitive cause of death. How are these two competitive? They are co-existent findings.\nIn the conclusion part, they opined the cause of death was blunt force trauma, which resulted in a fracture of the base of the skull. The authors did not mention the brain, the meninges, and the spinal cord findings.\nThe cause of death in cases of traumatic injury to the brain(TBI) can not be given as \"base-of-skull fractures\". The findings of the intracranial and vertebro-spinal structures are missing from the case report.\nThere is no mention of the reconstruction of the crime scene, and the authors did not support with solid evidence that the manner of death was suicidal.\nFurther, there are frequent grammatical errors throughout the manuscript. Many sentences require rephrasing.\n\nAdditionally, Â it should be explained why DNA samples were taken if the identity was already established. What is the practice at the author's place regarding the collection of DNA samples? If indicated, why were conventional samples like blood, molar tooth, and pieces of bone not preferred?\n\nIs the background of the caseâs history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-270
|
https://f1000research.com/articles/12-269/v1
|
13 Mar 23
|
{
"type": "Research Article",
"title": "Securitisation behind persona non grata:Â Implications to the theory and the cases regarding the Russian invasion of Ukraine in 2022",
"authors": [
"Yuki Moritani",
"Hajime Akiyama",
"Hajime Akiyama"
],
"abstract": "Background: Diplomats or consuls can be declared persona non grata (PNG) by receiving states. In many cases, it is declared for security reasons, claiming that issues caused by sending states are matters of national security of receiving states. The extant literature focused on legal aspects of it regarding diplomatic immunities and privileges, but the declarations of PNG in international affairs require considerations from security studies. Methods: Security is a social construct, and the process of construction is explained by securitisation theory by the Copenhagen School and the Paris School. The paper conducted an overview of PNG by a general examination of the declarations of PNG with securitisation theory. Further examinations were done focusing on series of the declarations of PNG during the Russian invasion of Ukraine in 2022. Along with the overall examination of PNG during the crisis, the analysis of the declaration of PNG to a Japanese Consul in Vladivostok, Russia was conducted as a unique case. Results: Generally, declaration of PNG is a result of securitisation within normal politics as the Paris School theorised, not exceeding normal politics as the Copenhagen School theorised. Also, two implications to the theory were found: 1) the need to focus on the existential threat; and 2) the need to consider a sphere of gradation between normal and emergency politics. The massacre in Bucha, Ukraine triggered many declarations of PNG, indicating that the cause of the securitisation of PNG was the massacre as an existential threat. The case of Vladivostok was a deviation from normal politics with a possible illegal detention of the consul, but the declaration of PNG itself is within normal politics. Rather than seeing the case simply as in the realm of emergency politics, it is better interpreted as a mixture of normal and emergency politics.",
"keywords": [
"Persona non grata",
"securitisation theory",
"the Copenhagen School",
"the Paris School",
"diplomatic privileges and immunities",
"Vienna Convention on Diplomatic Relations",
"Vienna Convention on Consular Relations",
"2022 Russian invasion of Ukraine"
],
"content": "Introduction\n\nDiplomacy is at the core of the practice of international relations. It is diplomacy that allows a state to deliver its intentions to other states and to make arrangements to form and sustain international relations.1 Thus, there is no reason not to study diplomatic issues from the research field of international relations. Basically, on a daily basis, diplomacy is done by diplomatic missions that are exchanged between states to sustain and develop relationships. Additionally, consular affairs have a significant role as well. To sustain diplomatic relations, privileges and immunities are given to diplomats and consuls.2 Such privileges and immunities should not be abused, but misuses can and do happen. When abused, persona non grata (PNG) can be declared by the receiving state to a diplomat or a consul from a sending state as an action to protect its state sovereignty. PNG was designed to be a significant system in international relations to sustain the diplomatic relations of states. However, not much academic attention has been given to PNG.\n\nPreviously, a small amount of research on PNG was mainly done in the field of international law. For those, PNG was an international legal issue regarding diplomatic privileges and immunities. However, its declarations are matters of international relations, not only international law. Usually, PNG has been issued for âan unfriendly attitude toward the (prospective) receiving state, violation of its laws or of international law, or improper diplomatic behavior or indiscretionsâ.3 The reason behind the declaration can be political, like âas a symbolic gesture as a way for a country to show displeasure with the actions of another country or entityâ.4 Also, it seems that â[t]he more the relation is fragile, and above all, based on conflicting interests, the more the states are not hesitating to Ref 5â declaring PNG. Such a tendency attracts consideration of PNG from the specific perspective of security. Security studies deal with not only military elements but with various other aspects including diplomacy.6 Moreover, as Eric Paul Witiw pointed out, PNG âallows the receiving state to prevent serious damage to its national securityâ.7\n\nThe declaration of PNG itself is not primarily a security policy. However, in many cases, receiving states announce that there was a security threat. The existence of such communication stimulates a hypothesis that the declaration of PNG is the result of securitisation. In particular, when PNG has been issued for security reasons, was PNG securitised? If so, in what sense? Mainly, this paper intends to make two contributions. One is to develop knowledge of PNG by approaching it from the perspectives of international relations and security. This contributes not only academically but also to develop understanding on current affairs. Such declarations of PNG can be found regarding the situation of the Russian invasion of Ukraine that broke out on 24 February 2022. Another contribution is the development of securitisation theory. By examining PNG with securitisation theory, the assumptions on the limitations of the current theory can be pointed out.\n\nThis paper is constructed with four sections excluding the introduction and conclusion. First, an overview of PNG from political and legal perspectives is provided. Next, a general examination of the declarations of PNG with securitisation theory is provided. Overall, this section shows that many declarations of PNG are results of securitisation within normal politics as the Paris School theorised, not exceeding normal politics as the Copenhagen School theorised. Also, two assumptions on securitisation theory, 1) the need to focus on the existential threat and 2) the need to consider a sphere of gradation between normal and emergency politics, are posed. To consider these assumptions, the paper focuses on PNG after the Russian invasion of Ukraine began for deeper analysis. In the third section, an overview of the declarations of PNG regarding the Russian invasion of Ukraine is given. The series of the declarations suggest the significance of the role of the existential threat to securitisation. In the fourth section, the case of the declaration of PNG to a Japanese consul in Vladivostok, Russia is analysed. This is a unique and significant case since not only the declaration of PNG but also the detention of a consul was seen. The case suggests the need to consider the situation as a mixture of normal and emergency politics, rather than simply labelling either normal or emergency politics.\n\n\nLaw and Politics of Persona Non Grata in Diplomacy\n\nThe practice similar to PNG is observable from ancient times, and it has been recognised in international law. PNG means â[p]erson not appreciatedâ in Latin,8 and it refers to âa diplomatic agent of a state is unacceptable to the receiving stateâ.9 It is understood that the concept of PNG was regarded as a customary law even before the conclusion of the Vienna Convention on Diplomatic Relations (VCDR) in 1961.10 Diplomacy is âthe management of international relations by negotiationâ.11 Distinguishing from foreign policy, Harold Nicolson pointed out the significance of negotiation in the concept of diplomacy saying the term âdiplomacyâ âdenotes the processes and machinery by which such negotiation is carried outâ.12 Since states indicate their willingness not to accept diplomats by declaring PNG, PNG makes it difficult to negotiate issues. In other words, PNG receives attention from the perspective of the effectiveness of diplomacy.\n\nAs a background, there are two different concepts related to PNG. On the one hand, there is a concept of sovereignty. Based on sovereignty, each state can decide which foreigners to allow or refuse the admission to the state, and preservation of security can be one function of sovereignty. On the other hand, there is sovereign immunity, and diplomats and consuls need to be regarded as representatives of the sovereign state, and as a result, they enjoy some kinds of privileges.13 Therefore, PNG can be regarded as a concept to coordinate these different concepts.14\n\nSovereign immunity has been recognised since the 19th Century. Since the beginning of the diplomatic system, when governments sent diplomatic representatives to other states throughout the world, sovereign immunity has been a formal one in diplomatic affairs. To sustain diplomatic relations, privileges and immunities are given to diplomats, so that diplomatic missions are exempt from the local legal forces fully. Embassies, where diplomatic missions are located, have territorial sovereignty of the sending state free from the law of the receiving state. The aim of diplomatic immunities is to allow states to maintain its works overseas in its embassies, which is necessary for the ambassador to âtrulyâ speak for the sovereign.15\n\nSuch immunities are also found with consuls. They are not entitled to diplomatic immunities since they are different from diplomats, but consuls also receive privileges and immunities. Moreover, practically, consuls receive more privileges and immunities than originally expected to maintain friendly relationships between states.16 As Luke T. Lee wrote, âthe existence of consular relations is the agreement to confer on consuls certain privileges and immunitiesâ.17 When these privileges and immunities by diplomats and consuls are abused, PNG can be declared by a receiving state to a diplomat or a consul from another state as an action to protect its state sovereignty.\n\nCurrently, there are two multilateral treaties that provide for PNG: the VCDR,18 which was adopted in 1961 and the Vienna Convention on Consular Relations (VCCR), which was adopted in 1963.19 Related to the case this paper covers, both Japan and Russia are contracting parties to these treaties. The scope of these treaties is different,20 but both of them provide for PNG. Article 9 of the VCDR provides that:\n\nâ1. The receiving State may at any time and without having to explain its decision, notify the sending State that the head of the mission or any member of the diplomatic staff of the mission is persona non grata or that any other member of the staff of the mission is not acceptable. In any such case, the sending State shall, as appropriate, either recall the person concerned or terminate his functions with the mission. A person may be declared non grata or not acceptable before arriving in the territory of the receiving State.\n\n2. If the sending State refuses or fails within a reasonable period to carry out its obligations under paragraph 1 of this article, the receiving State may refuse to recognize the person concerned as a member of the mission.â\n\nArticle 23 of the VCCR provides that:\n\nâ1. The receiving State may at any time notify the sending State that a consular officer is persona non grata or that any other member of the consular staff is not acceptable. In that event, the sending State shall, as the case may be, either recall the person concerned or terminate his functions with the consular post.\n\n2. If the sending State refuses or fails within a reasonable time to carry out its obligations under paragraph 1 of this article, the receiving State may, as the case may be, either withdraw the exequatur from the person concerned or cease to consider him as a member of the consular staff.\n\n3. A person appointed as a member of a consular post may be declared unacceptable before arriving in the territory of the receiving State or, if already in the receiving State, before entering on his duties with the consular post. In any such case, the sending State shall withdraw his appointment.\n\n4. In the cases mentioned in paragraphs 1 and 3 of this article, the receiving State is not obliged to give to the sending State reasons for its decision.â\n\nIt is important to note that neither of them obliges states to give reasons to declare PNG. During the negotiating history of the VCDR, there was not much discussion on the necessity to notify the reason of PNG because many states assumed that the reasons do not have to be shared.21\n\nAlthough there is no need to explain the reason for the declaration, it has been done with purpose and aim. The reason behind the declaration can be political, like âas a symbolic gesture as a way for a country to show displeasure with the actions of another country or entityâ22, as Nehaluddin Ahmad wrote. According to Amer Fakhoury, there were 10 types of the reason behind the declaration of PNG: 1) âviolating hosting stateâs lawsâ; 2) ânegative position of the sending states toward the interests of the hosting statesâ; 3) âpre-emptive action toward the sending stateâ; 4) âinterfering with its politics, or domestic affairsâ; 5) â[v]isiting sensitive areas in hosting countries without prior permissionâ; 6) âusing aggressive or undiplomatic language la[sic] against the exterior or interior hosting stateâs policiesâ; 7) âcriminal behavior either minor or majorâ; 8) âcriticizing its head of state or any high ranking personalityâ; 9) âoffenses against the security of the state such as espionage either political or economicâ; and 10) â[w]ar and severance of diplomatic relation between countries.â23\n\n\nSecuritisation and Persona Non Grata\n\nDiplomatic affairs can be a matter of security. It is true that the primary focus of security studies traditionally has been to explore âthe conditions that make the use of force more likely, the ways that the use of force affects individuals, states, and societies, and the specific policies that states adopt in order to prepare for, prevent, or engage in warâ.24 However, as Arnold Wolfers wrote, âthe term âsecurityâ covers a range of goals so wide that highly divergent policies can be interpreted as policies of securityâ.25 Stephen Walt also admitted that â[m]ilitary power is not the only source of national security, and military threats are not the only dangers that states faceâ.26\n\nEspecially with PNG, consideration from the perspective of security studies is necessary. PNG has been usually issued for âan unfriendly attitude toward the (prospective) receiving state, violation of its laws or of international law, or improper diplomatic behavior or indiscretionsâ.27 As already mentioned, Fakhoury introduced 10 types of the reason behind the declaration of PNG, and the last two reasons (âoffenses against the security of the state such as espionage either political or economicâ and â[w]ar and severance of diplomatic relation between countriesâ)28 indicate security is an important motivation to declare PNG. It is said that PNG was applied much during the Cold War mainly for spy activities âin a tit-for-tat fashionâ.29 Moreover, as Fakhoury pointed out, â[t]he more the relation is fragile, and above all, based on conflicting interests, the more the states are not hesitating toâ30 declaring PNG. This implies that international relations and security is relevant to the declaration of PNG. More importantly, the term ânational securityâ has been used in the announcements by governments for many cases of the declaration. Although PNG is originally a diplomatic issue, it is used as a security measure in many cases of the declaration. In other words, PNG is securitised.\n\nOle WÊver, the leading scholar of securitisation theory, argued that political leaders in a state declare an emergency condition claiming a need to use whatever means to deal with an issue by using the word âsecurity.â31 Extremely, it is possible to see anything such as environmental pollution, pandemic, abuse, or economic downturn as âsecurity threats.â However, seeing almost anything as a security issue âwould destroy its intellectual coherenceâ32 as Walt wrote. To define security coherently, along with Barry Buzan and Jaap de Wilde, WÊver argued that security is something different from the normal mode of politics. For WÊver, security is the emergence of âpolitics beyond the established rules of the gameâ33 by the issue being framed as âa special kind of politics or as above politicsâ.34 The theoretical school associated with this idea is called the Copenhagen School.\n\nThe Copenhagen School argues that âthe issue becomes a security issue - not necessarily because a real existential threat exists but because the issue is presented as such a threatâ.35 Based on this assumption, security is socially constructed with: 1) the securitising move, â[a] discourse that takes the form of presenting something as an existential threat to a referent objectâ36, by political leaders; and 2) the acceptance by audiences such as citizens, which provides legitimacy to security policy.37 Usually, policies exist because there is coercion and consent, according to the Copenhagen School. Rather than strictly requiring certain responses by audiences, Buzan, WÊver, and de Wilde wrote that the acceptance intends to check that securitising move gained âenough resonance for a platform to be made from which it is possible to legitimize emergency measuresâ.38\n\nRegarding emergency measures, the Copenhagen School makes a distinction between politics and security. According to the scholars of the Copenhagen School, any public issue can be located into three categorisations of spectrums: non-politicised, politicised, and securitised. Non-politicised issue is an issue that the state is not dealing with. Regarding an issue, no public debate is made and no political decision is made. When an issue turns into a matter of public policy that requires decision making and resource allocations by the government, it can be said that it is politicised. More importantly, the Copenhagen School sees that âarmed forces and intelligence services are carefully separated from normal political lifeâ39, especially in the Western developed and civilised political system.40 For the Copenhagen School, ideally, politics is something that can be done with routine procedures without any extraordinary measures. Thus, securitised issue is different from politicised issue since it is âpresented as an existential threat, requiring emergency measures and justifying actions outside the normal bounds of political procedureâ.41 Thus, for the Copenhagen School, securitisation is an extreme version of politicisation that exceeds from normal politics, that demands less democratic control over policies or even the legitimation of illegal government activities. The term âpanic politicsâ is also used to describe such a political situation.42 (See Table 1)\n\nFrom the approach of the Copenhagen School, the general cases of PNG cannot be said that it was securitised. As pointed out above, especially when PNG was declared for against hostile activities or severe inter-state relationship,43 the phrase ânational securityâ is used in the announcements by receiving states for many cases of the declaration of PNG. Receiving states show that what diplomats or consuls have done was threatening national security in the announcements. Such announcements are the securitising move that requires audiences to admit the necessity to deal with.44 With the legitimation of the declaration of PNG, it can be said that such securitising moves are accepted. However, more careful consideration is needed to argue that the declarations of PNG are the result of securitisation.\n\nAs the Copenhagen School noted, securitisation is fulfilled âby cases of existential threats that legitimize the breaking of rulesâ.45 It requires the measure to be done not in the arena of normal politics, but âoutside the normal bounds of political procedureâ.46 The declarations of PNG are legally admitted activities by states. Article 9 of the VCDR says that â[t]he receiving State may at any time and without having to explain its decision, notify the sending State that the head of the mission or any member of the diplomatic staff of the mission is persona non grata or that any other member of the staff of the mission is not acceptableâ.47 The declaration of PNG to consulars is also legal as Article 23 of the VCCR says: â[t]he receiving State may at any time notify the sending State that a consular office is persona non grata or that any other member of the consular staff is not acceptableâ.48 Since the declarations have clear legal basis, it can be said that the declarations of PNG are done within normal politics.\n\nHowever, although the usual cases of PNG are done in the arena of normal politics, it is still true that the declarations are done in the manner of security measures. This suggests that security policies can take place as a part of normal politics, although the Copenhagen School argued that âarmed forces and intelligence services are carefully separated from normal political lifeâ.49 This requires a different analysis from the Paris School, another school of securitisation theory.\n\nOne significant point regarding PNG is that the declaration is legally accepted even for security reasons. By criticising the theory by the Copenhagen School, the Paris School argued that there is a connectivity between normal politics and security. For example, although Thierry Balzacq shared a similar idea that securitisation is about governments taking extraordinary measures, he declined the idea to separate security and politics.50 Didier Bigo, a leading scholar of the Paris School, also pointed out that securitisation works continuously rather than being exceptional.51 For the Paris School, securitisation occurs within normal politics, not as the realm outside.52 (See Table 2)\n\nRather than focusing on the role of political discourse by political leaders in securitisation, the Paris School emphasises âthe day-to-day practices, of the bureaucracies that are necessary to understand how discourses work in practiceâ.53 Threats or risks are assessed and determined not only by the political leaders, but also by security practitioners and bureaucratic institutions. Thus, according to the Paris School, it is âthe role of the bureaucratic professionalization of the management of uneaseâ54 that should be taken into account in securitisation.\n\nIt is not only political elites as the Copenhagen School argued, but knowledge and technological resources that bureaucratic institutions and security professionals that âproduce a hierarchy of threatsâ.55 In other words, not necessarily by the political discourse but bureaucratic works can determine what threat and security is. Bureaucratic institutions and professionals make security legitimate by shaping political labels with manpower, time, and professional routines.56 Thus, the Paris School claims the necessity of the analysis of âthe internal logic of the field of professionals in the management of uneaseâ57 in the process of securitisation. (See Table 3)\n\nOne significant aspect of the internal logic for bureaucracy and professionals in the legal basis. Usually, the works by bureaucrats and professionals in agencies or institutions are done legally based on law.58 Regarding the declaration of PNG, Article 9 of the VCDR and Article 23 of the VCCR are the legal basis as seen above. Both articles allow a receiving state to declare PNG without explaining the reason for it.59 Thus, referring to the Paris School, it can be argued that securitisation of PNG in many cases is done within normal politics with legal basis even for security and emergency reasons.\n\n\nPersona Non Grata and the Russian Invasion of Ukraine\n\nAccording to the general examination, many cases of the declarations of PNG are the result of securitisation as seen above. Such can be seen in the situation regarding the Russian invasion of Ukraine in 2022. According to Foreign Policy, at least 394 Russian officials were expelled by the Western states by April 2022 after the Russian invasion of Ukraine began.60 The US Embassy and Consulates indicated that 19 states expelled Russian diplomats.61 The first case of the declaration of PNG was by the United States. On 28 February 2022, the United States announced that 12 Russian diplomats in the mission to the United Nations (UN) are PNG. According to the announcement, Russian diplomats were engaged in espionage activities in the United States. The United States mission to the United Nations (UN) mentioned that such espionage activities were âadverse to [American] national securityâ.62 Richard Mills, the Deputy Representative of the United States to the UN, stated that the decision of the declaration of PNG was made âso [Russian diplomats] do not harm the national security of the host countryâ.63 This case also can be analysed that the declaration was the result of securitisation within normal politics.\n\nSince the beginning of the Russian invasion of Ukraine, many cases of the expulsion of diplomats, consuls and diplomatic staff can be found. Below, Table 4, is the list of expulsions, including the declarations of PNG:64\n\nSome states indicate their intention behind the declaration of PNG and expulsion in their official statements. For example, on the Danish decision to expel 15 Russian intelligence officers who worked at the Russian embassy on 5 April, Jeppe Kofod, the Foreign Minister of Denmark, stated that: âThey pose a threat to our national security. Itâs in our mutual interests to maintain diplomatic ties, but we will not accept Russian espionage on Danish soilâ.101 What this indicated was that security is regarded as one important reason for the expulsion.\n\nAs can be seen from the list, many cases of the expulsion and the declaration of PNG can be found in the early April of 2022.102 Focusing on speech acts, as written above, governments claimed that the decisions were made due to âspying or ânational security reasonsââ103 by Russia. Other than examples above, José Manuel Albares, the Foreign Minister of Spain, announced that Russian âdiplomats and staff posed a âthreat to the interest of the countryââ.104 Italy also claimed that its decision was made due to national security reasons.105 However, the VCDR and the VCCR allow states to declare PNG âat any timeâ.106 Why did many states declare PNG in April 2022?\n\nThe possible reason behind the expulsions in early April was the massacre in Bucha. After the withdrawal of Russian forces from Bucha at the end of March, more than 400 dead bodies of citizens were found as this was reported in early April.107 As can be seen from the announcements of governments, decisions to expel and declare PNG to Russian diplomats and consuls were made as a response to the massacre.108 For example, Spain referred to the massacre as a reason for the expulsion.109 Foreign Minister Albares stated in the announcement that â[t]he unbearable images we have seen of the massacre of civilians in the town of Bucha after the withdrawal of the Russian army deeply outrage usâ.110 Germany argued that the massacre was a war crime, and the decision was made âas an immediate responseâ111 to the massacre in Bucha. The Foreign Minister of Lithuania stated that:\n\nâWhat the world has seen in Bucha, unfortunately, may only be the beginning. With other liberated cities, we may see more horrific examples of war crimes. All war crimes and crimes against humanity committed by the Russian armed forces in Ukraine will not be forgottenâ.112\n\nThe Foreign Ministry of Slovenia also âexpressed âthe strongest protestâ against the killings of civilians by Russian forces in Bucha and other townsâ.113 These cases indicate that the Russian invasion to Ukraine is the direct reason for expulsion rhetorically. It also indicates that the massacre in Bucha was one significant concern for states. This explains the timing of the expulsion and the declaration of PNG.114\n\nIn explaining the process of securitisation, securitisation theory does not emphasise the role of the existential threat itself. What the Copenhagen School focuses on as a significant producer of securitisation is political discourses. Buzan, WÊver and de Wilde clearly wrote that âthe issue becomes a security issue - not necessarily because a real existential threat exists but because the issue is presented as such a threatâ.115 Similarly, the Paris School also relatively undermined the effect of the existential threat itself but focuses more on the significance of professional internal logics to securitise an unease.116\n\nGenerally, as Fakhoury pointed out, the declarations of PNG can be found when the relationship between states is fragile.117 Considering the cases of PNG regarding the Russian invasion of Ukraine, the significant reason for securitisation seems to be the massacre in Bucha, which is the actual event. It is true that such decision making on the declaration of PNG is decided by political leaders or bureaucratic professionals. Although a widely shared concept or belief can be socially constructed by political elites or professional logic, âthere must also be a basis upon which the people can accept such a conceptâ.118 If so, objective character or degree of existential threat seems to matter in the establishment of securitisation significantly. What the examination of PNG with securitisation suggests is that more serious consideration of the role of existential threats is needed. (See Table 5)\n\n\nThe Detention of a Japanese Consul in Vladivostok and the Declaration of Persona Non Grata\n\nAs examined above, many cases of the declaration of PNG regarding the Russian invasion of Ukraine in 2022 were securitised within normal politics. However, there was one case that seems to be securitised in the sense of theory of the Copenhagen School. On 26 September 2022, the Ministry of Foreign Affairs of Russia told the Japanese Embassy in Russia that the activity of a Japanese consul in Vladivostok damaged Russian security interests and declared PNG to the consul. What makes this case notable is that the Japanese consul was physically detained along with the declaration of PNG. On the same day, the Federal Security Service of the Russian Federation (FSB) announced that it detained the Japanese consul in Vladivostok. It was claimed that the consul has done an activity of espionage, obtaining classified information illegally.119\n\nThe Japanese government claimed that the action which the Russian government pointed out was not present. The MOFA of Japan announced that âthere is no fact that the consular conducted an illegal activity as Russia allegesâ.120 To be noted, the determination of facts related to espionage is difficult from an academic point of view. It is almost impossible to confirm which claim is true. However, it is possible to examine the process of securitisation without considering the preciseness of espionage activities. As the Copenhagen School pointed out theoretically, the significant aim of securitisation is âto understand the processes of constructing a shared understanding of what is to be considered and collectively responded to as a threatâ.121 What is important here is the fact that the Russian government claimed a security reason to detain a Japanese consul and to declare PNG.\n\nThe Copenhagen School uses the term âpanic politicsâ for the securitised political situation.122 Securitisation in the sense of the Copenhagen School will allow a securitising actor to be free from rules or legal process that should bound policymakers normally.123 Panic politics is a situation âwhere departures from the rules of normal politics justify secrecy, additional executive powers, and activities that would otherwise be illegalâ.124 As examples of extraordinary means, the Copenhagen School listed âthe form of secrecy, levying taxes or conscription, [and] placing limitations on a specific taskâ.125 In this case, an extraordinary action that seems to be taken was an illegal activity.\n\nAs laws regarding this case, not only the consideration of the VCCR, but also the Consular Convention between Japan and the Union of Soviet Socialist Republics (Japan-Soviet Consular Convention) is needed. Both conventions provide rules on dealing with consuls judicially, but the rule is stricter in the VCCR than the Japan-Soviet Consular Convention. According to Article 41 of the VCCR, basically, â[c]onsular officers shall not be liable to arrest or detention pending trialâ.126 If the case is not âa grave crime and pursuant to a decision by the competent judicial authorityâ127, âconsular officers shall not be committed to prison or be liable to any other form of restriction on their personal freedom save in execution of a judicial decision of final effectâ.128 From this perspective, the detention of a Japanese consul in Vladivostok is problematic. Illegal espionage activities can be seen as a âgrave crime,â but âa decision by the competent judicial authorityâ129 is needed. If circumstances meet the condition above, âit has become necessary to detain a consular officerâ130 as the Article stated. However, a decision was not made by a judicial authority, but by the FSB. With this, the detention of a Japanese consul by Russia can be possibly illegal, against Article 41 of the VCCR. From this, it can be argued that the case was the matter of panic politics as the Copenhagen School conceptualised.\n\nThe general examinations of PNG with securitisation supported the theoretical argument of the Paris School, not security as the deviation from normal politics but security within normal politics.131 However, this specific case of PNG associated with the detention of a consul suggests that securitisation of PNG can result in exceeding normal politics. Although the Paris School has not dealt seriously with security policy outside normal politics, this requires the consideration that security policy actually can step into the realm of panic politics.\n\nWith further consideration, the case suggests that the Copenhagen School also has a problem, especially its clear distinction of normal politics and the realm of emergency politics. In theory, extraordinary means are simply and equally considered as elements of panic politics. However, with the consideration of the Japan-Soviet Consular Convention, it cannot be argued that the case was âfullyâ panic politics. The Japan-Soviet Consular Convention, which was adopted in 1966 and is currently effective between Japan and Russia, admits judicial immunity to consuls. Article 18 of the Convention provides that â[a] state shall enjoy immunity from the jurisdiction of the receiving state with respect to official actionâ.132 If an action by a Japanese consul the Russian government accused as an espionage activity was an âofficial action,â an activity of a consul had to be outside of the jurisdiction of Russia. As mentioned above, it is almost impossible to find out if an espionage activity was done by a Japanese consul as an official action. If not, the application of the Russian jurisdiction to a Japanese consul can be possible.\n\nIn total, the Russian detention of a Japanese consul in Vladivostok can possibly be seen as an illegal activity. It is difficult to legally justify the Russian action considering Article 41 of the VCCR. However, this does not mean that Russia broke international law overall regarding this case. Depending on the situation there is a possibility to find a certain legal basis in Article 18 of the Japan-Soviet Consular Convention.133 Moreover, it must be noted that the declaration of PNG itself has a legal basis in Article 23 of the VCCR. The declaration of PNG with the reasoning of an espionage activity itself is legal as Article 23 of the VCCR states: â[t]he receiving State may at any time notify the sending State that a consular officer is persona non grataâ134 and âis not obliged to give to the sending State reasons for its decisionâ.135\n\nThere are both legal and illegal perspectives to examine this issue. With this situation, it is too simple to label the status as âpanic politicsâ. It seems more precise to find the gradation between normal politics and panic politics, and the case fits the interpretation that stepped into the realm of panic politics but not fully. This provides another implication to securitisation theory, the need to consider the gradation between normal politics and the realm of emergency politics.\n\n\nConclusion\n\nAcademically, PNG was mainly dealt with as an issue of international law regarding diplomatic immunities and privileges. Although the declaration of PNG itself is not primarily a security policy, many cases of it are associated with the claims that the issues states are facing are matters of national security. Security is a social construct. The construction of security can be explained by securitisation of the Copenhagen School and the Paris School. This paper provided two main contributions. One is the development of the knowledge of PNG by approaching from international relations and security. By generally examining PNG with securitisation theory, it can be argued that many declarations of PNG are results of securitisation within normal politics as the Paris School theorised. This contributes not only academically but also to develop understanding on current affairs. Such declarations of PNG can be found regarding the situation of the Russian invasion of Ukraine that broke out on 24 February 2022.\n\nWith the consideration of the declarations of PNG regarding the situation of the Russian invasion of Ukraine, implications to the development of securitisation theory were found as another contribution of this paper. One is the need for securitisation theory to be more aware of the role of the existential threat in the securitising process. Considering the cases of the declaration of PNG in 2022, many declarations were made after the unravelling of the massacre of Bucha. It should be taken into account that threats and risks are not only made by political leaders or security professionals, but also with an actual basis that people can accept. Another theoretical implication is the need to consider the gradation between normal politics and the realm of panic politics. The specific case of the Russian declaration of PNG to a Japanese consul in Vladivostok after the detention was the result of securitisation as what the Copenhagen School theorised. However, although it was possibly illegal in total, some other legal perspectives can be found. Although the case seems to have stepped into the realm of panic politics, it is not fully panic politics. Thus, rather than simply labeling the status as âpanic politics,â it seems more precise to find the gradation between normal politics and panic politics theoretically.\n\nThis complicated legality/illegality is interesting from a legal perspective. There is no single unified legal norm, but there are legal norms which are different in scope. As a result, there can be confusion in interpreting the legality of any given action. This paper clarifies that how to evaluate diverse legal norms is one important area of study, and this also indicates the link between law, politics and diplomacy.\n\nHowever, there are some limitations of this paper. First, in the examination of the diplomatic relations regarding the Russian invasion of Ukraine in 2022, this paper did not distinguish the practice of expulsion and the declaration of PNG in Table 4. The authors assume that both have many things in common in practice and it was difficult to distinguish in reality, so that both of them were covered in this paper. However, it must be noted that they are different in theory, and close analysis of each concept and reality will be required in the further research. Second, although the examinations of the declarations of PNG after the unravelling of the massacre in Bucha and the case of Vladivostok were done, the degree of analysis is limited due to the lack of access to internal policy making process. However, the strength of the examination is the attempt to see the result of policy and its relations of discourse and legal basis. Lastly, although the theoretical implications to securitisation theory were made, the number of cases dealt in this paper is limited. By using the current issue as the cases, this paper can contribute academically and socially, but more considerations of the actual cases are needed for the further development of theory. In the future, these limitations need to be overcome.\n\nIn recent years, many scholars and policymakers have been saying that the international order is facing a crisis. Such a trend seems to continue. When the tension rises in international relations, possibilities of the declaration of PNG can rise. The declaration can also raise the tension of international affairs. The analysis of the declaration of PNG from the perspectives of international relations and security studies is necessary to develop better understanding on international affairs. This paper showed the potential of the analysis with securitisation theory. This is only a first step for future research, encouraging the further development of the field.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgement\n\nThe authors express the deepest gratitude and respect to Mr Shinya Oguma (Research Fellow, Policy Research Department, National Institute for Defense Studies) for his significant comments and advice on the research.\n\n\nReferences\n\nAFP: EU allies expel more than 200 Russian diplomats and staff amid outrage over Bucha killings. 5 Apr. 22. 2022a. 6 Dec. 22.Reference Source\n\nAFP: Russia summons Luxembourg envoy over ambassador expulsion. 19 Apr. 22.2022b. 28 Nov. 22.Reference Source\n\nAhmad N: The obligation of diplomats to respect the laws and regulations of the hosting state: A critical overview of the international practices. 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Vienna Conventions on Diplomatic and Consular Relations. New York:Carnegie Endowment for International Peace;1969; 41â76.\n\nMaÄek SR: Slovenia expels 33 Russian diplomats, EURACTIV. 6 Apr. 22. 2022. 27 Nov. 22.Reference Source\n\nMinistry of Foreign Affairs of Japan: Expulsion of diplomats and officials from the Embassy of the Russian Federation and the Trade Representation of the Russian Federation in Japan. 8 Apr. 22.2022a. 28 Nov. 22.Reference Source\n\nMinistry of Foreign Affairs of Japan: Vice-Minister for Foreign Affairs Mr. MORI summons Mr. Mikhail Y. GALUZIN, Ambassador of the Russian Federation to Japan. 4 Oct. 22.2022b. 8 Dec. 22.Reference Source\n\nMinistry of Foreign Affairs of the Republic of Lithuania: Lithuania sends Russian ambassador away in response to Russian crimes in Ukraine. 5 Apr. 22.2022. 3 Dec. 22. Reference Source\n\nNakanishi H, Ishida A, Tadokoro M: Kokusaiseijigaku [International politics: Theories and perspectives]. Tokyo:YÅ«hikaku;2015.\n\nNeagu B: Romania expels 10 Russian diplomats, EURACTIV, 6 Apr. 22. 2022. 28 Nov. 22.Reference Source\n\nNHK: Amerika seifu Rosia Kokuren DaihyÅ no gaikÅkan 12 nin no kokugai tsuihÅ wo kettei [US government decided to expel 12 Russian diplomats in the Permanent Mission to the UN]. 1 Mar. 22.2022. 28 Nov. 22.Reference Source\n\nNichols M: U.S. expels 12 Russian U.N. diplomats over security concerns, Reuters. 1 Mar. 22.2022. 27 Nov. 22.Reference Source\n\nNicolson SH: Diplomacy. Washington DC:Institute for the Study of Diplomacy;1988.\n\nNikkei: Rosia, urajiosutoku no Nihon sÅryÅjikanin wo ichiji kÅsoku [Russia temporarily detained a Japanese consul in Vladivostok]. 27 Sep. 22.2022. 26 Oct. 22.Reference Source\n\nNoyan O: Germany expels 40 Russian diplomats in response to massacre in Ukraine, EURACTIV, 5 Apr. 22. 2022. 28 Nov. 22.Reference Source\n\nNV: Russian diplomat expelled from Moldova after missile incident. 1 Nov. 22.2022. 27 Nov. 22.Reference Source\n\nOzturk T: Montenegro declares 9 Russian diplomats persona non grata, Anadolu Agency, 29 Sep. 22. 2022. 27 Nov. 22.Reference Source\n\nOzturk T, Abay EG: Russia declares 14 Bulgarian diplomatic staff persona non grata, Anadolu Agency, 5 Aug. 22. 2022. 27 Nov. 22.Reference Source\n\nQuann J: Two Irish diplomats âasked to leaveâ Moscow, Dent. News, 7 Apr. 22. 2022. 27 Nov. 22.Reference Source\n\nRadio Free Europe/Radio Liberty: Russia expels Romanian diplomat in response to âunfriendlyâ state. 8 Sep. 22.2022. 27 Nov. 22.Reference Source\n\nRedazione ANSA: Ukraine: Italy has expelled 30 Russian diplomats-Di Maio (3). 5 Apr. 22.2022. 3 Dec.22.Reference Source\n\nReuters: Denmark expels 15 Russian diplomats; Moscow to retaliate. 5 Apr. 22.2022a. 28 Nov. 22.Reference Source\n\nReuters: Lithuania expels Russian ambassador over civilian deaths near Kyiv. 5 Apr. 22.2022b. 27 Nov. 22.Reference Source\n\nReuters: Montenegro declares Russian diplomat persona non grata. 4 Mar. 22.2022c. 27 Nov. 22.Reference Source\n\nReuters: Russia moves to expel U.S. diplomats in tit-for-tat move. 24 Mar. 22.2022d. 27 Nov. 22.Reference Source\n\nReuters: Spain to expel around 25 Russian diplomats, foreign minister says. 5 Apr. 22.2022e. 28 Nov. 22.Reference Source\n\nReuters: Sweden joins European nations in expelling Russian diplomats. 5 Apr. 22.2022f. 28 Nov. 22.Reference Source\n\nReuters Editor: Rosia, Nihon gaikÅkan 8 nin tsuihÅ e hÅfuku sochi [Russia expels 8 Japanese diplomats as a retaliation]. 28 Apr. 22.2022. 28 Nov. 22.Reference Source\n\nRFE/RL: Russia expels five Croatian diplomats over âunfriendly actionsâ, RadioFreeEurope/RadioLiberty . 27 May. 22 2022. 27 Nov. 22.Reference Source\n\nRoe P: Is securitization a ânegativeâ concept? Revisiting the normative debate over normal versus extraordinary politics. Secur. Dialogue. 2012; 43(3): 249â266.\n\nRudenko O: Hundreds of murdered civilians discovered as Russians withdraw from towns near Kyiv (GRAPHIC IMAGES), The Kyiv Independent, 3 Apr. 22. 2022. 3 Dec. 22.Reference Source\n\nTeslova E: Lithuania declares Russian embassy employee persona non grata, Anadolu Agency, 1 Dec. 22. 2022. 3 Dec. 22.Reference Source\n\nTHE ASAHI SHIMBUN: Japan plans to expel Russian diplomat in tit-for-tat move. 4 Oct. 22.2022. 27 Nov. 22.Reference Source\n\nThe Yomiuri Shimbun: Japan retaliates by designating Russian consul persona non grata. 4 Oct. 22. 2022. 28 Nov. 22.Reference Source\n\nUnited States Mission to the United Nations: Statement on the decision to initiate the expulsion of Russian diplomats in New York. 2 Feb. 22.2022. 27 Dec. 22.Reference Source\n\nU.S. Embassy & Consulates in Italy: Countries expel Russian diplomats in protest over Ukraine. 1 May. 22.2022. 3 Dec. 22.Reference Source\n\nVakil C: Russia declares 45 Polish diplomats âpersona non grataâ in diplomatic expulsion tit for tat, The Hill. 8 Apr. 22. 2022. 3 Dec. 22.Reference Source\n\nVelazquez D, Lambert Y: Luxembourg expels Russian diplomat, Luxembourg Times. 6 Apr. 22. 2022. 28 Nov. 22.Reference Source\n\nWalt SM: The renaissance of security studies. Int. Stud. Q. 1991; 35(2): 211â239.\n\nWÊver O: Identity, integration and security: Solving the sovereignty puzzle in E.U. studies. J. Int. Aff. 1995; 48(2): 389â431.\n\nWestfall S, Simon MF: Which countries have expelled Russian diplomats over Ukraine? Wash. Post, 25 Apr. 22. 2022. 3 Dec. 22.Reference Source\n\nWitiw EP: Persona non grata: Expelling diplomats who abuse their privileges. New York Law School Journal of International and Comparative Law. 1988; 9(2&3): 345â359.\n\nWolfers A: National securityâ as an ambiguous symbol. Political Science Quarterly. 1952; 67(4): 481â502. Publisher Full Text\n\n\nFootnotes\n\n1 See Nakanishi et al., 2015, 123; Grieco et al., 2019, 240.\n\n2 See Grieco et al., 2019, 241-242; Lee, 1969, 58-59.\n\n3 Fakhoury, 2017, 116.\n\n4 Ahmad, 2020, 2.\n\n5 Fakhoury, 2017, 117.\n\n6 Walt, 1991, 213.\n\n7 Witiw, 1988, 359.\n\n8 Ahmad, 2020, 3.\n\n9 Bledsoe and Boczek, 1987, 112.\n\n10 Ahmad, 2020, 4.\n\n11 Nicolson, 1988, 4.\n\n12 Ibid., 3-4.\n\n13 Preamble of the VCDR provides that âthe purpose of such privileges and immunities is not to benefit individuals but to ensure the efficient performance of the functions of diplomatic missions as representing States.â\n\n14 Ahmad, (2020, 4) calls PNG as âbalance, worthiness, and justice between the principle of sovereignty and territorial jurisdiction on one hand, and the principle of inviolability and immunity on the other hand.â\n\n15 Grieco et al., 2019, 241-242.\n\n16 Lee, 1969, 59.\n\n17 Ibid., 58-59.\n\n18 Adopted 18 April 1961, entered into force 24 April 1964, United Nations Treaty Series, vol. 500, p. 95.\n\n19 Adopted 24 April 1963, entered into force 19 March 1967, United Nations Treaty Series, vol. 596, p. 261.\n\n20 The VCDR covers the diplomats (see Article 1 of the VCDR), and the VCCR covers âa consular officerâ (Article 1, the VCCR).\n\n21 Denza 1998, 62.\n\n22 Ahmad 2020, 2.\n\n23 Fakhoury 2017, 116-117.\n\n24 Walt 1991, 212.\n\n25 Wolfers 1952, 484.\n\n26 Walt 1991, 213.\n\n27 Fakhoury 2017, 116.\n\n28 Ibid., 116-117.\n\n29 Ahmad 2020, 2. See also Crawford 2008, 401; Lee 1969, 58-59.\n\n30 Fakhoury 2017, 117.\n\n31 See WÊver 1995, 405.\n\n32 Walt 1991, 213.\n\n33 Buzan et al., 1998, 23.\n\n34 Ibid.\n\n35 Ibid., 24.\n\n36 Ibid., 25.\n\n37 Ibid., 23-25; Buzan 1997, 14.\n\n38 Buzan et al. 1998, 25.\n\n39 Ibid., 28.\n\n40 Howell and Richter-Montpetit 2020, 8.\n\n41 Buzan et al. 1998, 24.\n\n42 Ibid., 24-29; Buzan 1997, 14.\n\n43 Fakhoury 2017, 116-117.\n\n44 Buzan 1997, 14; Buzan et al. 1998, 23-25.\n\n45 Buzan et al. 1998, 25.\n\n46 Ibid., 24.\n\n47 Article 9, the VCDR.\n\n48 Article 23, the VCCR.\n\n49 Buzan et al. 1998, 28.\n\n50 Balzacq 2019, 343.\n\n51 Bigo 2002, 73.\n\n52 See Balzacq 2019; Bigo 2002; Kamino 2021, 31-51; Kudo 2011, 187-189.\n\n53 Bigo 2002, 73.\n\n54 Ibid., 74.\n\n55 Ibid., 76.\n\n56 Ibid., 75.\n\n57 Ibid., 86.\n\n58 As the other example of a research focusing on the significance of legal basis as the internal logic for bureaucracy and professionals in the process of securitisation, see a conference presentation: Moritani, Yuki. 2021. âSakerarenai anzenhoshÅka?: COVID-19 to Jieitai no katsuyÅ [Unavoidable securitization?: COVID-19 and the usage of the Self-Defense Forces],â in the 14th Seminar, Japan Association for International Security.\n\n59 Article 9, the VCDR; Article 23, the VCCR.\n\n60 Gramer and Yang 2022.\n\n61 Namely, Austria, Bulgaria, Czech Republic, Denmark, Estonia, Finland, Germany, Greece, Ireland, Italy, Japan, Latvia, Lithuania, Norway, Poland, Romania, Slovenia, Spain, Sweden. U.S. Embassy & Consulates in Italy 2022.\n\n62 United States Mission to the United Nations 2022.\n\n63 Quoted in Nichols 2022.\n\n64 Note that the below is not necessarily a full list of cases of expulsion and declaration of PNG. They are the cases that the authors are aware of based on their research.\n\n65 NHK 2022; Nichols 2022.\n\n66 Reuters 2022c.\n\n67 Westfall and Simon 2022.\n\n68 Al Jazeera 2022a.\n\n69 Reuters 2022d.\n\n70 Westfall and Simon 2022.\n\n71 Ibid.\n\n72 EURACTIV.com with Reuters 2022.\n\n73 Westfall and Simon 2022.\n\n74 Reuters 2022b.\n\n75 Al Jazeera 2022b; Kazmin et al. 2022; Noyan 2022.\n\n76 EEAS Press Team 2022.\n\n77 ERR News 2022; Kazmin et al. 2022; MaÄek 2022; Neagu 2022; Reuters 2022a; Reuters 2022e; Reuters 2022f.\n\n78 Al Jazeera 2022c; Velazquez and Lambert 2022.\n\n79 Aydogan 2022.\n\n80 Quann 2022.\n\n81 Konno 2022; Ministry of Foreign Affairs of Japan 2022a.\n\n82 Vakil 2022.\n\n83 DAILY SABAH WITH AGENCIES 2022.\n\n84 AFP 2022b; Gijs 2022.\n\n85 Dynes 2022.\n\n86 Westfall and Simon 2022.\n\n87 Reuters Editor 2022.\n\n88 Basso et al. 2022.\n\n89 RFE/RL 2022.\n\n90 Ozturk and Abay 2022.\n\n91 Ibid.\n\n92 Interfax 2022.\n\n93 Radio Free Europe/Radio Liberty 2022.\n\n94 THE ASAHI SHIMBUN 2022; The Yomiuri Shimbun 2022.\n\n95 Ozturk 2022.\n\n96 JakuÄionis 2022.\n\n97 The Yomiuri Shimbun 2022.\n\n98 Chirciu 2022.\n\n99 NV 2022.\n\n100 Teslova 2022.\n\n101 https://twitter.com/JeppeKofod/status/1511262192217300992(5 Apr. 22) accessed on 3 Dec. 22.\n\n102 It must be noted that not all expulsions were associated with the declaration of PNG.\n\n103 AFP 2022a.\n\n104 Connolly 2022.\n\n105 AFP 2022a; Redazione ANSA 2022.\n\n106 Article 9, the VCDR; Article 23, the VCCR.\n\n107 For instance, the Kyiv Independent reported the massacre on 3 April 2022. Rudenko, 2022.\n\n108 AFP 2022a; Connolly 2020.\n\n109 Westfall and Simon 2022.\n\n110 Quoted in AFP 2022a.\n\n111 Connolly 2022.\n\n112 Ministry of Foreign Affairs of the Republic of Lithuania 2022.\n\n113 MaÄek 2022.\n\n114 However, it should be noted that some states like Montenegro and Slovakia took their actions in early March, which might have different reasons.\n\n115 Buzan et al. 1998, 24.\n\n116 Bigo 2002, 73-76.\n\n117 Fakhoury 2017, 117.\n\n118 Akiyama 2019, Note 55.\n\n119 See Nikkei 2022.\n\n120 MOFA 2022b.\n\n121 Buzan et al., 1998, 26.\n\n122 See Buzan, 1997, 14; Roe, 2012, 249-266.\n\n123 Buzan et al., 1998, 25.\n\n124 Buzan, 1997, 14.\n\n125 Buzan et al., 1998, 24.\n\n126 Article 41, the VCCR.\n\n127 Ibid.\n\n128 Ibid.\n\n129 Ibid.\n\n130 Ibid.\n\n131 Bigo, 2002, 73.\n\n132 Article 18, the Japan-Soviet Consular Convention.\n\n133 However, as the common understanding, Japan lacks the capability of espionage and intelligence abroad. See Kotani 2014, 56-57.\n\n134 Article 23, the VCCR.\n\n135 Ibid."
}
|
[
{
"id": "196135",
"date": "23 Aug 2023",
"name": "Dionysios STIVAS",
"expertise": [
"Reviewer Expertise Security",
"Securitization",
"Internationa Relations",
"International Law."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper constitutes an interesting approach to declarations of PNG observing them from the perspectives of the securitization theory. The main argument is that declaring PNG in the context of the war in Ukraine constitutes securitization from the perspective of the Paris School but not from that of the Copenhagen School except from one instance: the PNG and detention of the Japanese Consul to Vladivostok.\nThe paper would benefit if the authors conducted a more extensive literature review on the emergency measures/actions and the Copenhagen School. This is because the authors rely extensively on the (important admittedly) book of Buzan, Waever, and De Wilde from 1998. However, lots have been written by other authors on this matter in the last 25 years. The Copenhagen School theory has been updated and most of the criticisms of the theory have been addressed. An update of the Paris School theory with more recent literature would also be desirable.\nWhat is more, the authors are advised to merge Table 3 with Table 5 as they look as repeating each other. Tables 2 and 6 need more details within the text of the tables or in the notes of the tables. As they stand now, it is difficult for the reader to translate the tables. Lastly, the paper would benefit from additional proofreading of the last 3 paragraphs of the last section and the entire Conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "196149",
"date": "24 Aug 2023",
"name": "Didem Aydindag",
"expertise": [
"Reviewer Expertise Security Studies",
"Critical Security Studies",
"IR Theory",
"Middle Eastern Politics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is very interesting and the topic is up to date, however the part regarding Paris School of securitization needs to be more developed. For example from pages 5-7 a detailed understanding of Copenhagen School is given whereas for Paris School section only 2-3 paragraphs are given and there is a sharp shift from the argument flow. It is not very clear why a shift to Paris School was necessary in the argument or to what extent it is necessary.\nThe author can a) either improve the section regarding the connection between the case and the Paris School or b) focus on more recent arguments within the Copenhagen School and explore PNG from second and third generation Copenhagen School studies even including Ole Waever's more recent articles as well.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-269
|
https://f1000research.com/articles/11-1149/v1
|
07 Oct 22
|
{
"type": "Brief Report",
"title": "Sex-related differences in symptom presentation of patients with aneurysmal subarachnoid hemorrhage",
"authors": [
"Laura Philine Westphal",
"Stefan Yu Bögli",
"Jana Werner",
"Francesca Casagrande",
"Emanuela Keller",
"Giovanna Brandi",
"Stefan Yu Bögli",
"Jana Werner",
"Francesca Casagrande",
"Emanuela Keller",
"Giovanna Brandi"
],
"abstract": "Background: In patients with myocardial infarction, atypical symptoms at onset have been demonstrated in women. We aimed to investigate the presence of sex-related differences in symptom presentation in patients with aneurysmal subarachnoid hemorrhage (aSAH) to enable earlier diagnosis and treatment. Methods: We assessed symptoms on admission to hospital in 343 patients with aSAH in this retrospective single-center cohort-study. Univariate statistical analysis was performed by comparing sexes including the whole study population and subgroups (dichotomized using Fisher scale 1-2 vs. 3-4, WFNS grade 1-3 vs. 4-5, and anterior vs. posterior circulation aneurysms, respectively). Results: The majority of patients was female (63.6%, n=218, vs. 36.4%, n=125), the mean age 57.4 years (standard deviation (SD) 13.3) with older women compared to men (59.2, SD 13.8, vs. 54.4, SD 11.6; p=0.003). Anterior communicating artery (AcomA) aneurysms were most common (30.9%, n=106), predominantly in men (43.2%, n=54, vs. 23.9%, n=52; p=0.0002), whereas posterior communicating artery (PcomA) aneurysms were more frequent in women (19.3%, n=42, vs. 8.8%, n=11; p=0.005). Exercise-induced headache was more often reported by men (10.4%, n=13, vs. 5%, n=11; p=0.04) in all patients as well as in the subgroup of WFNS 1-3. Anisocoria was more frequent in women within the subgroup of severely impaired consciousness (WFNS 4-5; 25.3%, n=22, vs. 10.7%, n=6; p=0.032). For all other symptoms, there was no evidence for sex-specific differences in the whole study group as well as in subgroups. Conclusion: Our results show no evidence for relevant sex-related differences in symptom presentation at onset in aSAH patients. Women presenting with an acute onset anisocoria should be screened even more carefully for an underlying ruptured Pcom aneurysm.",
"keywords": [
"aneurysmal subarachnoid hemorrhage",
"symptom presentation",
"gender medicine",
"sex-related differences",
"women"
],
"content": "Introduction\n\nAneurysmal subarachnoid hemorrhage (aSAH) accounts for 5% of all strokes and for 75-80% of spontaneous SAH with an overall incidence of 6-8/100.000 people per year in Western populations.1,2 As aSAH occurs at a younger age and has a high case fatality, the loss of productive life years ranges on the same level as the one from cerebral infarction.2,3 Therefore, the assessment and accurate interpretation of clinical symptoms including the knowledge of possible sex-related differences in patients with aSAH is a key factor for rapid and correct diagnosis as well as further monitoring and treatment decisions.\n\nIn cardiovascular disease as myocardial infarction (MI), several studies demonstrated that chest pain is the most common symptom in males, while women more frequently suffered from atypical or unspecific associated symptoms such as dizziness, nausea, palpitations and pain or discomfort in the jaw, neck, arms or between the shoulder blades resulting in higher rates of unrecognized MI.4,5 This sex-related difference in symptom presentation does not only contribute to lower rates of diagnosis of MI in women, but also to delayed or prevented treatment leading to worsened outcome in women compared to men.5â8\n\nRegarding sex-related differences in aSAH patients, it is known that aSAH affects more women than men2,9 and women have a higher risk of death from SAH increasing with age.10 Moreover, ruptured aneurysms in women are mainly found in the internal carotid artery (ICA), whereas in men in the anterior cerebral artery (ACA).11 However, sex-related differences in symptom presentation have not been examined in-depth in this patient group so far.\n\nWe aimed to investigate if symptom presentation at onset in patients with aSAH differed between men and women after correction for possible confounders as aneurysm location and severity of hemorrhage. Sex-related atypical or different symptoms could thereby identify patients at higher risk for delayed diagnosis, which could lead to an increased focus on more diverse symptoms and the need for a more detailed anamnesis at symptom onset.\n\n\nMethods\n\nIn this retrospective single-center cohort-study, we screened 370 and enrolled 343 consecutive patients with acute aSAH admitted to the Neurocritical Care Unit (NCCU) of the University Hospital Zurich, Switzerland, as a tertiary care center between January 2016 and December 2021. Patients were also included when they first presented in a primary or secondary care center with following transfer to the NCCU of the University Hospital Zurich. Patients were excluded when presenting with additional other acute cerebrovascular diseases (n=2) or confounding sources of bleeding (n=1) in order to provide a homogenous patient cohort as well as when a written informed consent was missing (n=24), see Figure 1 for a flow chart of the study population. aSAH was diagnosed by radiological findings on computed tomography (CT) and after evidence of a ruptured aneurysm on CT-angiography (CTA) scans on admission or subsequent digital substraction angiography (DSA). Patients with aSAH included intracranial aneurysms of the anterior and posterior circulation as well as all severity levels defined by the Fisher grading scale12 and all degrees of clinical impairment defined by the World Federation of Neurosurgical Societies (WFNS) scale.13\n\nWritten informed consent was obtained in all patients included in the study or next of kin. The study was performed according to the ethical guidelines of the Canton of Zurich with approval of the local ethics committee of Zurich (KEK: 2022-00270).\n\nOn admission to hospital, we assessed demographic and clinical data, comorbidities presented by the Charlson comorbidity index (CCI) and radiologic findings as the aneurysm location, the presence of hydrocephalus defined as a relative bicaudate index > 1.6 on CT scan, intraventricular hemorrhage or additional intracerebral hemorrhage.14,15 The severity of aSAH based on radiologic findings was quantified using the Fisher score, the decline of consciousness and severity of neurological impairment was graded by the WFNS grade. Initial GCS was defined as the first documented score by the emergency service, primary, secondary or tertiary care centers, whereas GCS on admission was the first documented score on admission to the University Hospital Zurich. Outcome was classified by the Glasgow coma scale-extended (GOSE) assessed by physicians of the department of neurosurgery at the University Hospital Zurich during follow-up at three months after onset of bleeding.\n\nDescriptive or categorical variables are reported as counts/percentages, continuous variables as mean ± standard deviation or as median including the interquartile range (IQR) as appropriate. For the analysis of associated factors, patient characteristics and presented symptoms of patients were dichotomized by sex. For subgroup analyses, patients dichotomized by sex were stratified by the Fisher scale, WFNS grade and aneurysm location, respectively. We tested all continuous data for normal distribution using the Shapiro-Wilk's test. Categorical variables were compared with Pearson's Ï2 or Fisher's exact test, continuous variables using the t-test or MannâWhitney U test for parametric and non-parametric data, respectively, where appropriate. There were no missing values regarding the variables of clinical symptom presentation and there was no loss to follow-up at three months after onset of aSAH. P-values â€0.05 were considered to be statistically significant. All calculations were performed using SPSS version 26.\n\n\nResults\n\nWe screened 370 patients with aSAH and included 343 into the analysis. A detailed flow chart of the patients included in the study is provided in Figure 1. For all patients a follow-up at three months after onset of aSAH could be assessed or was classified accordingly if patients died within the follow-up period. There were no missing data for the assessed clinical variables.\n\nAs presented in Table 1, the majority of patients was female (n=218, 63.6%). Women were older than men (59.2, SD 13.8, vs. 54.4, SD 11.6; p=0.003). Most aneurysms were located in the anterior circulation (79%, n=217) with the anterior communicating artery (AcomA) being the most common aneurysm location (30.9%, n=106) predominantly in men (43.2%, n=54, vs. 23.9%, n=52; p=0.0002), whereas aneurysms of the posterior communicating artery (PcomA) were more frequent in women (19.3%, n=42, vs. 8.8%, n=11; p=0.005). 70.5% (n=241) of all patients demonstrated an intraventricular hemorrhage (IVH) at onset with no evidence of a sex difference (p=0.81).\n\n* Observed or suspected.\n\n** Systolic blood pressure >200 mmHg.\n\nThe median initial Glasgow Coma Scale (GCS) as well as the GCS on admission within all patients was 14 (interquartile range (IQR) 8-15), which did not differ between the two sexes. We provide a detailed sex-related list of all symptoms assessed at SAH onset in Table 1.\n\nAmong 46.6% (n=160) of all patients a loss of consciousness occurred during SAH onset, 43.1% (n=148) presented with an observed or suspected seizure and only 21.3% (n=73) with a focal neurological deficit on admission with a trend towards more women concerned (23.9%, n=52, vs. 16.8%, n=21; p=0.25). 69.7% (n=239) of all patients presented with headache and among these 56% (n=192) with acute onset headache and 23.9% (n=82) with persistent headache with a mean duration of 4.13 days (SD 7.0) without evidence for a sex-related difference. Exercise-induced headache was the only symptom with a statistically significant sex-related difference occurring more often within men (10.4%, n=13, vs. 5%, n=11; p=0.04).\n\nWe additionally performed a subgroup analysis stratified by Fisher scale as a parameter of radiologically defined severity of aSAH. Patients were classified into two groups with Fisher scale 1-2 (n=37) and Fisher scale 3-4 (n=305). When analyzing the sex-related symptom presentation separately within these two groups, there was no evidence for a different clinical presentation between sexes at SAH onset.\n\nIn a further subgroup analysis stratified by WFNS grade as a marker of clinical severity based on the GCS on admission, patients were classified into the groups WFNS 1-3 (n=200) defined as GCS 13-15 and WFNS 4-5 (n=143) defined as patients with a GCS †12. Within the subgroup of WFNS 1-3, men presented again more often with exercise-induced headache at SAH onset compared to women (18.8%, n=13, vs. 6.1%, n=8; p=0.007). In the subgroup of WFNS 3-4 with severely impaired consciousness, anisocoria revealed to occur statistically significantly more often among women (25.3%, n=22, vs. 10.7%, n=6; p=0.032), see Table 2.\n\nWhen analyzing the subgroups of anterior (n=271) and posterior circulation aneurysms (n=72), there was no evidence for sex-related differences in symptom presentation at SAH onset. Only meningism tended to occur more often in women with aneurysms of the anterior circulation, but without significance (18.9%, n=32, vs. 8.8%, n=9; p=0.064), see Table 3.\n\n\nDiscussion\n\nPatients with aSAH represent a severely ill patient group with often devastating outcome.16,17 Earliest possible detection and treatment of the ruptured aneurysm is imperative for the improvement of outcome.17 In patients with aSAH, differences in clinical and subjective symptoms at onset between both sexes have not been studied in-depth so far with only few studies addressing symptoms of aSAH patients in general without differentiating between sexes.18 Our primary hypothesis was that similarly to the findings in cardiovascular disease, symptom presentation in female aSAH patients might also differ.\n\nIn patients with MI, women often present with atypical symptoms as dizziness, nausea or extracardiac pain at onset compared to men resulting in lower rates of detection, treatment and worsened outcome in women.5â8,12\n\nIn our study population, we found that only exercise-induced headache was more frequently reported by men compared to women. For all other clinical symptoms at aSAH onset, there was no evidence for differentiating symptoms with respect to sex. To reduce the bias that symptoms could not be assessed correctly due to limitations to get the medical history in patients with impaired consciousness, we conducted a subgroup analysis considering patients with only slight to modest decline in consciousness (WFNS 1-3). In this subgroup, the findings did not change thereby supporting and validating in a first step our results from the whole study group. Regarding patients with a severe decline in consciousness, only anisocoria occurred more frequently within female patients (WFNS 4-5; p=0.032). In line with that, ruptured Pcom aneurysms were found more frequently in women (p=0.005), with this type of aneurysm representing a common cause of acute oculomotor nerve palsy resulting in anisocoria.19 Accordingly, women with acute onset anisocoria should be screened even more carefully for an underlying ruptured PcomA aneurysm.\n\nIn our study, we detected only few differing symptoms at onset with respect to sex in aSAH patients compared to patients with MI, which might be explained by the fact that the epicardial innervation is dermatoma-specific enabling a vast range of different, atypical or extracardiac symptoms. In contrast, the intracranial innervation is not, thus symptoms in aSAH patients are mainly stereotype with headache (69.7%), nausea or vomiting (54.8%) and decrease of consciousness (63%) when intracranial pressure rises due to local or generalized space-occupying effects or acute hydrocephalus after aSAH or consist of focal neurological deficits depending on the aneurysm location.\n\nIn previous aSAH patient cohorts, women were more often affected compared to men,2,9 which is in line with our findings. Humoral factors as decreased estrogen levels after menopause contribute to an increased risk of aneurysm formation in older females.20,21 Interestingly, female sex has been associated with an increased risk of aneurysm formation, but not with an increased risk of aneurysm rupture.22 Aneurysm location has been reported to differ between both sexes with more ruptured aneurysms of the ACA in men,11 which could be confirmed in our study with predominantly ruptured AcomA aneurysm in men. In women the ICA has been described as the most affected vessel of ruptured aneurysms,11 whereas we detected most frequently MCA aneurysms in women. However, PcomA aneurysms were found significantly more often in women compared to men.\n\nThe strength of our study is that we provide a broad overview of possible clinical symptoms in aSAH patients within a large and homogenous patient cohort (Table 1), thereby offering a clinical and practice-oriented guide with the aim of rapid and accurate diagnosis and treatment. We also performed a subgroup analysis taking into account the Fisher scale, WFNS grade and aneurysm location to rule out a possible bias due to limited assessment of symptoms at onset in patients with impaired consciousness (WFNS 4-5). Limitations of the study are firstly the single-center and retrospective type of study possibly leading to a certain bias in comparability and generalization of study results. Furthermore, a validation cohort with larger patient numbers should be investigated to confirm our results. In addition, due to the high amount of decreased vigilance (63%) in aSAH patients, the case history often had to be taken by a third party, which might result in a certain inaccuracy of the assessed symptoms at onset. To rule out that bias, we performed a subgroup analysis adjusted for the severity of hemorrhage (Fisher scale) and the level of consciousness (WFNS grade), respectively. By demonstrating that there was again no evidence for gender-related differing symptoms in patients with only slight to moderate decrease in vigilance (WFNS 1-3), the results of the whole study group were confirmed.\n\nIn conclusion, our results show no evidence for relevant sex-related differences in symptom presentation at onset in aSAH patients with stable findings when only analyzing patients with slight to moderate decrease in consciousness (WFNS 1-3). Further studies with comparative patient cohorts preferably with aSAH, but also other acute intracranial processes as hemorrhagic or ischemic stroke are needed to validate these results and provide a better understanding of possible sex related differences and specific treatment options in these patients.\n\n\nAuthor contributions\n\nLaura P. Westphal conducted data processing and interpretation and wrote the first draft of the manuscript. Stefan Y. Bögli was involved in data collection and performed the statistical analysis. Jana Werner and Francesca Casagrande were involved in data collection and critically revised the manuscript. Emanuela Keller critically revised the manuscript. Giovanna Brandi designed the study, was involved in data analysis and critically revised the manuscript. All authors contributed to manuscript revision and approved the final version of the manuscript.\n\n\nData availability\n\nThe original data set can be found on Zenodo. Sex-related differences in symptom presentation of patients with aneurysmal subarachnoid hemorrhage. DOI: https://doi.org/10.5281/zenodo.7007417.23\n\nThis project contains the following underlying data:\n\n- Retrospective study regarding sex-related differences in symptom presentation of patients with aneurysmal subarachnoid hemorrhage.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe study was reported according to the STROBE guidelines for observational studies.24\n\nZenodo. Completed STROBE guidelines checklist for observational studies of the manuscript âSex-related differences in symptom presentation of patients with aneurysmal subarachnoid hemorrhageâ. DOI: https://doi.org/10.5281/zenodo.7102315.25",
"appendix": "References\n\nGreenberg M: Handbook of neurosurgery. 6th ed.New York:Thieme;2006.\n\nde Rooij NK , Linn FH, van der Plas JA , et al.: Incidence of subarachnoid haemorrhage: a systematic review with emphasis on region, age, gender and time trends. J. Neurol. Neurosurg. Psychiatry. 2007; 78(12): 1365â1372. PubMed Abstract | Publisher Full Text\n\nJohnston SC, Selvin S, Gress DR: The burden, trends, and demographics of mortality from subarachnoid hemorrhage. Neurology. 1998 May; 50(5): 1413â1418. PubMed Abstract | Publisher Full Text\n\nvan der Ende MY , Juarez-Orozco LE, Waardenburg I, et al.: Sex-Based Differences in Unrecognized Myocardial Infarction. J. Am. Heart Assoc. 2020; 9(13): e015519. PubMed Abstract | Publisher Full Text\n\nLichtman JH, Leifheit EC, Safdar B, et al.: Sex Differences in the Presentation and Perception of Symptoms Among Young Patients With Myocardial Infarction: Evidence from the VIRGO Study (Variation in Recovery: Role of Gender on Outcomes of Young AMI Patients). Circulation. 2018; 137(8): 781â790. PubMed Abstract | Publisher Full Text\n\nWenger NK: Women and coronary heart disease: a century after Herrick: understudied, underdiagnosed, and undertreated. Circulation. 2012; 126: 604â611. PubMed Abstract | Publisher Full Text\n\nMehta L, Beckie T, Devon H, et al.: Acute myocardial infarction in women: a scientific statement from the American Heart Association. Circulation. 2016; 133: 916â947. Publisher Full Text\n\nMadonis SM, Skelding KA, Roberts M: Management of acute coronary syndromes: special considerations in women. Heart. 2017; 103: 1638â1646. PubMed Abstract | Publisher Full Text\n\nWermer MJ, van der Schaaf IC , Algra A, et al.: Risk of rupture of unruptured intracranial aneurysms in relation to patient and aneurysm characteristics: an updated meta-analysis. Stroke. 2007 Apr; 38(4): 1404â1410. PubMed Abstract | Publisher Full Text\n\nAyala C, Croft JB, Greenlund KJ, et al.: Sex differences in US mortality rates for stroke and stroke subtypes by race/ethnicity and age, 1995-1998. Stroke. 2002; 33: 1197â1201. PubMed Abstract | Publisher Full Text\n\nGhods AJ, Lopes D, Chen M: Gender differences in cerebral aneurysm location. Front. Neurol. 2012; 3(78). Published 2012 May 21. PubMed Abstract | Publisher Full Text\n\nFisher CM, Kistler JP, Davis JM: Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery. 1980 Jan; 6(1): 1â9. PubMed Abstract | Publisher Full Text\n\nDrake CG, Hunt WE, Sano K, et al.: Report of World Federation of Neurological Surgeons Committee on a Universal Subarachnoid Hemorrhage Grading Scale. J. Neurosurg. 1988 Jun; 68(6): 985â986. Publisher Full Text\n\nCharlson ME, Pompei P, Ales KL, et al.: A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J. Chronic Dis. 1987; 40(5): 373â383. PubMed Abstract | Publisher Full Text\n\nvan Gijn J , Hijdra A, Wijdicks EF, et al.: Acute hydrocephalus after aneurysmal subarachnoid hemorrhage. J. Neurosurg. 1985 Sep; 63(3): 355â362. Publisher Full Text\n\nHop JW, Rinkel GJ, Algra A, et al.: Case-fatality rates and functional outcome after subarachnoid hemorrhage: a systematic review. Stroke. 1997 Mar; 28(3): 660â664. Publisher Full Text\n\nZacharia BE, Hickman ZL, Grobelny BT, et al.: Epidemiology of aneurysmal subarachnoid hemorrhage. Neurosurg. Clin. N. Am. 2010 Apr; 21(2): 221â233. Publisher Full Text\n\nLinn FH, Rinkel GJ, Algra A, et al.: Headache characteristics in subarachnoid haemorrhage and benign thunderclap headache. J. Neurol. Neurosurg. Psychiatry. 1998; 65(5): 791â793. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSoni SR: Aneurysms of the posterior communicating artery and oculomotor paresis. J. Neurol. Neurosurg. Psychiatry. 1974; 37(4): 475â484. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTada Y, Makino H, Furukawa H, et al.: Roles of estrogen in the formation of intracranial aneurysms in ovariectomized female mice. Neurosurgery. 2014; 75: 690â695. PubMed Abstract | Publisher Full Text\n\nTabuchi S: Relationship between postmenopausal estrogen deficiency and aneurysmal subarachnoid hemorrhage. Behav. Neurol. 2015; 2015: 1â6. Publisher Full Text\n\nGreving JP, Wermer MJ, Brown RD, et al.: Development of the PHASES score for prediction of risk of rupture of intracranial aneurysms: a pooled analysis of six prospective cohort studies. Lancet Neurol. 2014; 13: 59â66. PubMed Abstract | Publisher Full Text\n\nWestphal LP, Bögli SY, Werner J, et al.: Sex-related differences in symptom presentation of patients with aneurysmal subarachnoid hemorrhage [Data set]. Zenodo. 2022. Publisher Full Text\n\nvon Elm E , Altman DG, Egger M, et al.: Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. BMJ. 2007; 335: 806â808. PubMed Abstract | Publisher Full Text\n\nWestphal LP, Bögli SY, Werner J, et al.: Completed STROBE guidelines checklist for observational studies of the manuscript âSex-related differences in symptom presentation of patients with aneurysmal subarachnoid hemorrhageâ. Zenodo. 2022. Publisher Full Text"
}
|
[
{
"id": "162644",
"date": "07 Mar 2023",
"name": "Matthias Haenggi",
"expertise": [
"Reviewer Expertise Critical care medicine"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDr Westphal and her colleagues aimed to answer the question if there are any differences in symptom presentation for aSAH with regard to sex, similar to different symptom presentation between women and men with ACS/MI. Although there are few distinctions in baseline characteristics and aneurysm location, symptoms at onset of SAH did not differ between women and men. This holds true after adjusting for severity and aneurysm location.\nThe manuscript is clear, concise and well-written/easy to understand. The conclusions drawn are based on the results. The limits are outlined, but I am missing one or two sentences about the troubles frequently occurring if trial results are âneutralâ or ânegativeâ: for theoretical reasons, failure to prove a difference does not imply that there is no difference (âabsence of evidence is not evidence of absenceâ). It can simply occur if the number of events is low, as it has happened here in subgroup analysis, or the total n are low. It is the clinical context which is important, not the p-value. Therefore I am not convinced that âa validation cohort with larger patient numbers should be investigated to confirm our resultsâ, as stated by the authors. Variation of symptoms in presentation is high, so knowledge of a statistically significant sex difference in a symptom cannot rule in/rule out aSAH.\nThere are some minor issues:\nFisher score WFNS grade/GOSE: for the readers who do not remember the scoring systems, please present in a few words the grading (the higher the score, the worseâŠ)\n\nTable 1: \"WLST\" is not defined, I assume 'withdrawal of life support therapies'\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9438",
"date": "03 Apr 2023",
"name": "Laura Westphal",
"role": "Author Response",
"response": "We thank the reviewer for the precise and valuable review of the article. We have now completed an updated version of the manuscript, in which we adapted and included the suggested points according to the review. In detail, we included in the conclusion part at the end of the discussion that, due to smaller patient numbers in subgroup analyses, the absence of evidence for relevant sex-related differences in symptom presentation of patients with aSAH does not imply that there are no such differences. We additionally adapted the sentence that our results need to be validated in the way that results need to be verified and confirmed in further studies in the future with larger patient cohorts. Furthermore, we thank the reviewer for the important aspect to explain in more detail the Fisher, WFNS and GOS-E scale used for clinical and diagnostic classification of patients with aSAH. The according changes can be found in the methods section. Additionally, we added a list of abbreviations at the end of table 1 in order to explain all abbreviations used in the table, including WLST for \"Withdrawal of life-supporting treatment\". We hope that we could address all mentioned points of the review within the adapted manuscript, which is now available on the website."
}
]
}
] | 1
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https://f1000research.com/articles/11-1149
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https://f1000research.com/articles/11-933/v1
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15 Aug 22
|
{
"type": "Brief Report",
"title": "Enhancement of the activity of the antimicrobial peptides HNP1 and LL-37 by bovine pancreatic ribonuclease A",
"authors": [
"Bryan Ericksen"
],
"abstract": "Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula.\n\nMethods: The virtual colony count (VCC) microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification.\n\nResults: Adding tRNA 1:1 to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x105 CFU/mL did not enhance activity. Increasing the inoculum to 6.25x107 CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA.\n\nConclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations and biofilms, conditions where the antimicrobial agent alone is relatively ineffective.",
"keywords": [
"antimicrobial peptide",
"ribonuclease"
],
"content": "Introduction\n\nAlthough cationic antimicrobial peptides (CAPs) have been studied as possible therapeutic agents for many years, few have survived clinical trials to become useful antibiotics (Mishra et al. 2017). Three CAPs are representative of three different structural classes that contribute to the human innate immune system: human neutrophil peptide 1 (HNP1), an alpha defensin; human beta defensin 1 (HBD1); and the human cathelicidin LL-37 (De Smet 2005). One reason why CAP drug candidates have failed to gain approval is a lack of efficacy (Magana 2020). I demonstrated a pronounced inoculum effect when the defensin HNP1 was assayed against high inocula of Escherichia coli ATCC 25922, such that the antimicrobial peptide almost completely lost activity under those conditions (Ericksen 2020). A pronounced inoculum effect was also observed when HNP1 was assayed against Staphylococcus aureus ATCC 29213 and Bacillus cereus ATCC 10876. What might cause this decrease in efficacy at high cell concentrations? The molecular basis of the inoculum effect is unclear. However, one possibility is that bacterial cells produce defensin inhibitors that are at a higher concentration when there are more cells present.\n\nOne possible type of inhibition is that polyanions might bind and inhibit CAPs by electrostatic attraction. Here I hypothesize that the polyanion tRNA might inhibit CAPs, that inhibition by RNA is partially responsible for the inoculum effect, and that the addition of ribonuclease could enhance antimicrobial peptide activity, restoring some of the efficacy lost at high cell concentrations.\n\n\nMethods\n\nThe VCC assay was adapted for high inocula as previously described (Ericksen 2020), and volumes were adjusted to allow for the addition of yeast tRNA (Sigma from Saccharomyces cerevisiae) and/or bovine pancreatic ribonuclease (Roche or Macherey-Nagel (MN)). HNP1, LL-37 and HBD1 were synthesized ABI 433A synthesizer using an optimized HBTU activation/DIEA in situ neutralization protocol developed by Kent and coworkers for Boc chemistry solid phase peptide synthesis as previously described (Zhao 2013; Pazgier 2013; Bharucha 2021). Two inocula were studied: the standard inoculum of 5Ã105 CFU/mL, with cells from a seed culture diluted in 10 mM sodium phosphate pH 7.4, and a high inoculum of 6.25Ã107 CFU/mL, equivalent to adding undiluted seed culture. Antimicrobial peptides were incubated in 10 mM sodium phosphate pH 7.4 plus 1% tryptic soy broth (TSB) for two hours at 37°C shaking every 5 minutes for 3 seconds in a Tecan Infinite M1000 plate reader. An equal volume of twice-concentrated Mueller Hinton Broth was then added and 96-well plates were read for 12 hours in the plate reader and then some wells containing cell clumps were photographed under 10x magnification. In one experiment, the concentration of TSB present in phosphate buffer was adjusted.\n\n\nResults\n\nAdding tRNA 1:1 to HNP1 at the standard inoculum almost completely abrogated activity (Figure 1). Adding Roche RNase 1:1 to HNP1 at the standard inoculum of 5Ã105 CFU/mL did not enhance activity. Increasing the inoculum to 6.25Ã107 CFU/mL almost abrogated HNP1 activity (Figure 2). However, adding RNase 25:1 to HNP1 enhanced activity at the high inoculum. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA (Figure 3). At the high inoculum, LL-37 was enhanced, but LL-37 showed greater activity than HNP1 in the absence of RNase (Figure 4). HBD1 activity was not enhanced by RNase. RNase was not active in the absence of antimicrobial peptides. The observations with HNP1 at the high inolculum were repeated using a second RNase manufacturer, Macherey-Nagel (Figure 5). The experiment with MN RNase was repeated (Figure 6). 1% TSB was used in most assays, but the %TSB was varied in one experiment, resulting in maximum activity at 4% TSB with either 5à or 25à MN RNase added (Figure 7). Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides with or without RNase and at the standard inoculum in the presence of HNP1+tRNA (Figure 8). The VCC assays were conducted with TSB added to the 10 mM sodium phosphate incubation buffer. The enhancement of activity caused by RNase was observed with LL-37 but not HNP1 when washed cells were used, indicating that RNase operates by different mechanisms with the two antimicrobial peptides. Although biofilm formation was not directly assayed, it is assumed that the cell clumps photographed at 10x magnification are biofilms. Ribonuclease did not enhance HBD1 activity at the 6.25Ã107 CFU/mL inoculum, demonstrating a strong inoculum effect with HBD1 vs. E. coli. LL-37 had a much lesser inoculum effect against E. coli. The effect of ribonuclease on HNP1 is strongest with lowest amounts of TSB present in the phosphate buffer during the 2 hour incubation. The ability of tRNA to abrogate HNP1 and HBD1 activity, and the failure of tRNA to affect LL-37 activity, at the standard inoculum cannot be explained by net charge. Possibly, hydrophobic interactions play a role in tRNA binding and inhibition. It is also possible that tRNA inducing biofilm formation impacts HNP1 and HBD1 more than LL-37.\n\nActivity with HNP1 and both tRNA and the highest concentration of RNase was essentially the same as HNP1 plus RNase alone, indicating the enhancement of activity overcomes inhibition by tRNA. RNase in the absence of antimicrobial peptides was not antimicrobial.\n\nHBD1 was assayed at the standard inoculum in the presence of 1:1 tRNA. Two preparations of HNP1 were assayed in the absence of tRNA as positive controls.\n\nHBD1 was assayed at the high inoculum with RNase.\n\nLeft panel: 128 ÎŒg/mL HNP1 at the high inoculum. Right panel: 128 ÎŒg/mL HNP1 + 1:5 RNase at the high inoculum.\n\n\nConclusions\n\nAntimicrobial assays are ordinarily conducted using a single antimicrobial agent, studying its effect in isolation. However, the experiments presented here may offer a glimpse into a more realistic in vivo scenario, in which multiple antimicrobial agents work in concert against infection. Eight RNases are encoded by the human genome, many of which have potent antimicrobial activity, such as RNase 7 expressed in epithelial cells (Sorrentino 2010). Bovine pancreatic RNase A, on the other hand, has a digestive function degrading RNA and an antimicrobial function has not normally been ascribed to it. RNase A is a basic protein (pI = 9.63). It is unknown whether the RNA-degrading activity of RNase or its cationicity is responsible for the enhancement of HNP1 and LL-37 activity. Product literature suggests assaying RNase A using 100 mM Tris buffer, pH 7.4. Enzymatic activity in 10 mM sodium phosphate buffer was not tested, but RNase A is very stable with four disulfide bonds.\n\nThe variation in the amount of TSB present in 10 mM phosphate buffer revealed that the increase in activity caused by a small amount of nutrients present, allowing some growth during the two-hour incubation, is counterbalanced by the inhibition of defensin activity at higher TSB concentrations, presumably by the salt content of TSB. This same effect is probably partially responsible for the almost complete abrogation of activity of HNP1 when undiluted seed culture is added to the 96-well plate at the high inoculum in the absence of RNase, since the salt concentration is much higher than in assays at the standard inoculum where the seed culture is diluted in 10 mM sodium phosphate buffer before adding to the 96-well plate.\n\nThe vast majority of published VCC assays were conducted at the standard inoculum, reflecting a general reliance on the standard inoculum in a wide range of published antimicrobial assays. Under these conditions, cells are predominantly planktonic. However, a high inoculum may be more medically relevant, since high cell concentrations and biofilms can accompany acute infections. This study demonstrates the utility of conducting assays at a high inoculum, revealing details of antimicrobial activity that would be missed if the antimicrobial agents were studied only at the standard inoculum. Further studies using animal models are necessary to determine whether the enhancement of activity observed at the high inoculum is sufficient to enable the infected host to overcome bacterial infections.\n\nIt should be emphasized that both RNA and ribonucleases are ubiquitous in vivo. Therefore, these experiments may be more biologically relevant than VCC experiments lacking RNA or ribonuclease. There are several possible sources of bacterial RNA that might be present at the site of a bacterial infection. Firstly, bacteria normally secrete RNA during their growth, which may have a role in the extracellular matrix of biofilms (Ozoline 2019). The results of the experiments presented here suggest that this secreted RNA may also be a bacterial defense mechanism against antimicrobial peptides. Secondly, once antimicrobial peptides are released at the infection site, cell lysis may result in the release of intracellular RNAs, including mRNA and tRNA. Thirdly, host RNA may be present. Therefore, inhibition by RNA must be regarded as a common obstacle to effective antimicrobial activity that frequently occurs in real world scenarios.\n\nThe combination of an antimicrobial peptide with a ribonuclease could be regarded as a novel invention that could possibly be used as a therapy to treat bacterial infections. LL-37 and RNase 1 have been shown to act synergistically to kill E. coli (Eller 2020). RNases have been tested in clinical trials as chemotherapeutics for the treatment of cancer (Ardelt 2009).\n\nFurther studies are warranted to determine whether these results could be generalized to antimicrobial peptide-nuclease combinations, as might be suggested by the presence of DNA in biofilms. A combination of an antimicrobial peptide with both deoxyribonuclease (DNase) and RNase might be expected to be more potent than the combination of the antimicrobial peptide and RNase in the absence of DNase, because DNA is considered a more prevalent structural component of biofilms than RNA (Gilan 2013). DNase is an approved drug, dornase alfa (Pulmozyme), which cuts apart extracellular DNA in the lungs of cystic fibrosis patients, making the mucus thinner and easier to expel (Wagener 2012). It is possible that DNase in combination with an antimicrobial peptide and RNase would form an effective treatment against acute bacterial infections. A new generation of antimicrobial peptide-nuclease combinations would offer a new hope that peptides that are sometimes defeated by the resistance mechanism of biofilm formation can be repurposed to degrade biofilms instead, with increased activity to fight infections.\n\n\nData availability\n\nFigshare: Enhancement of Antimicrobial Peptide Activity by Ribonuclease (virtual colony count data), https://doi.org/10.6084/m9.figshare.20352996.v1 (Ericksen 2022).\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgments\n\nI thank Wuyuan Lu for providing antimicrobial peptides and helpful discussions.\n\n\nReferences\n\nArdelt W, Ardelt B, Darzynkiewicz Z: Ribonucleases as potential modalities in anticancer therapy. Eur. J. Pharmacol. 2009; 625(1-3): 181â189. PubMed Abstract | Publisher Full Text\n\nBharucha JP, Sun L, Lu W, et al.: Human beta-defensin 2 and 3 Inhibit HIV-1 replication in macrophages. Front. Cell. Infect. Microbiol. 2021; 11: 535352. PubMed Abstract | Publisher Full Text\n\nDe Smet K, Contreras R: Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol. Lett. 2005; 27(18): 1337â1347. PubMed Abstract | Publisher Full Text\n\nEller CH, Raines RT: Antimicrobial synergy of a ribonuclease and a peptide secreted by human cells. ACS Infectious Diseases. 2020; 6(11): 3083â3088. PubMed Abstract | Publisher Full Text\n\nEricksen B: Virtual colony count. Wiklj. Sci. 2020; 3(1): 3. Publisher Full Text\n\nEricksen B: Enhancement of Antimicrobial Peptide Activity by Ribonuclease. Figshare. [Dataset].2022. Publisher Full Text\n\nGilan I, Sivan A: Extracellular DNA plays an important structural role in the biofilm of the plastic degrading actinomycete Rhodococcus ruber. Adv. Microbiol. 2013; 3(8): 40674.\n\nMagana M, Pushpanathan M, Santos AL, et al.: The value of antimicrobial peptides in the age of resistance. Lancet Infect. Dis. 2020; 20(9): e216âe230. PubMed Abstract | Publisher Full Text\n\nMishra B, Reiling S, Zarena D, et al.: Host defense antimicrobial peptides as antibiotics: design and application strategies. Curr. Opin. Chem. Biol. 2017; 38: 87â96. PubMed Abstract | Publisher Full Text\n\nOzoline O, Jass J: Editorial: secretion and signalling of bacterial RNAs. FEMS Microbiol. Lett. 2019; 366(1): fny281. Publisher Full Text\n\nPazgier M, Ericksen B, Ling M, et al.: Structural and functional analysis of the pro-domain of human cathelicidin, LL-37. Biochemistry. 2013; 52(9): 1547â1558. PubMed Abstract | Publisher Full Text\n\nSorrentino S: The eight human âcanonicalâ ribonucleases: Molecular diversity, catalytic properties, and special biological actions of the enzyme proteins. FEBS Lett. 2010; 584: 2194â2200. PubMed Abstract | Publisher Full Text\n\nWagener JS, Kupfer O: Dornase alfa (Pulmozyme). Curr. Opin. Pulm. Med. 2012; 18(6): 609â614. Publisher Full Text\n\nZhao L, Tolbert WD, Ericksen B, et al.: Single, double and quadruple alanine substitutions at oligomeric interfaces identify hydrophobicity as the key determinant of human neutrophil alpha defensin HNP1 function. PLoS One. 2013; 8(11): e78937. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "147551",
"date": "07 Sep 2022",
"name": "William R. Jacobs",
"expertise": [
"Reviewer Expertise Microbial genetics",
"tuberculosis",
"leprosy",
"herpes."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHi Dr. Erickson,\n\nThank you for sharing this research with us. The idea of using antimicrobial peptide-ribonuclease combinations could be beneficial for use when the microbial agent alone is ineffective, such as when used against high bacterial concentrations and biofilms. However, the virtual colony count has many caveats that would make the data not reflective of the true killing effect of the peptides. For this paper, why not simply use colony forming units? The author hypothesizes that higher densities of bacteria cause inactivation of the peptides by secretion of tRNAs, but provides no data substantiating this hypothesis. The author could have taken the supernatant fluid from the more densely populated culture and identified compounds that inactivated the peptides. An alternative hypothesis is that the bacterial cells are entering into a persistent state where they are expressing a genetic program that prevents killing by the peptides. Overall, the data is convincing that tRNA inhibits peptide activity, but itâs unclear that this has real world relevance. The author does show a modest effect of peptide-ribonuclease combinations on high inoculum cultures, but the VCC is an inadequate measure of killing. I believe that parts of this manuscript should be reworked and more data should be obtained to prove the effectiveness of these combinations.\nMinor Comments:\nIn the results section of the abstract I think the ordering could be more streamlined. Currently it goes through all the results for HNP1 and then goes through the HBD1 and LL-37 results together and I am not sure this is the most effective way to convey this information succinctly, especially seeing it is mostly pulled as written from the beginning of the results section. An alternative way would be to describe all three together.\n\nIn methods âHNP1, LL-37 and HBD1 were synthesized ABI 433A synthesizer using an optimizedâ may read better as âwere synthesized with an ABI 433A synthesizer.â\n\nIn some figures, especially 2, 5, and 6, the different shapes are difficult to parse. Thereâs a bunch of small, similar looking, overlapping shapes, and the way the shapes look on the key do not always perfectly correspond to the graph. For example, in Figure 2 in the key 25x RNase looks like a single uptick but Iâm pretty sure from looking at the graph section itâs actually a plus sign. I think that if you write the inoculum (with CFUs/mL not just high or standard) in the figure title you don't need it in the key. It just makes things more cluttered to look at and harder to read for the relevant information differentiating each line. If you disagree thatâs fine, but it should be consistent: Figure 1 doesnât have CFUs/mL in the key, Figure 2 does, Figure 3 does for some of the conditions, etc.\n\nFigure 3 is called âLL-37 was assayed at the standard inoculum with or without tRNA,â but this is also where you show your HBD1 tRNA data so the title should reflect that. Also you should show how effective HBD1 is before tRNA is used for inhibition especially since you say that tRNA is abrogating the HBD1 activity but Iâm not seeing what the activity looked like originally. I have a similar issue with Figure 4 where I think the HBD1 data without RNase should be given to match LL-37.\n\nYou write that a âpronounced inoculum effect was also observed when HNP1 was assayed against Staphylococcus aureus ATCC 29213 and Bacillus cereus ATCC 10876.â Do you believe that doing the RNase and tRNA experiments with these bacteria would show similar results?\n\nYou write that âthe enhancement of activity caused by RNase was observed with LL-37 but not HNP1 when washed cells were used, indicating that RNase operates by different mechanisms with the two antimicrobial peptidesâ I think you must mean HBD1 instead of HNP1 or what I am getting from your graphs is wildly different from what you are. Additionally, I donât see a difference between LL-37 alone vs LL-37 with 25x RNase in Figure 4 in anything besides the highest peptide condition, and thereâs nothing on any of your graphs that mark differences as statistically significant.\n\nWhat mechanism would you think the RNase improves LL-37 activity by since it is much less impacted by tRNA and high inoculum as the defensins so thereâs less of a problem for the RNase to solve. Additionally, it would be good to show RNase either reversing or failing to reverse the effect of tRNA on HBD1 like you do for HNP1, especially if you want to claim that RNase is not enhancing HBD1 activity.\n\nRegarding mechanism, has the author isolated mutants of the bacterial strains that are resistant to peptide function? A rigorous genetic analysis could provide important insights into the peptides specificities and functions.\n\nFurther, you use a log scale for experiments done at a standard inoculum, but not for those at a high inoculum, so it seems clear that even when using the RNase, these peptides are orders of magnitude less effective at high bacterial concentrations. Is a 30% decrease in virtual survival at the highest peptide concentration enough to consider these combinations effective when compared with non-CAP antibiotics?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "8749",
"date": "27 Sep 2022",
"name": "Bryan Ericksen",
"role": "Author Response",
"response": "Dear Dr. Jacobs, You have not provided any rational reason for your prejudice against the virtual colony count (VCC) assay. You suggest using the colony count assay, but VCC has one major advantage: in the colony count assay, cells must be diluted after exposure to antimicrobial agents in order to result in 30-300 colonies per plate. This dilution is problematic when cells are assayed at the standard inoculum, but much more so at a high inoculum. If an inoculum of 6.25x10^7 CFU/mL is used, and survival is .5, then cells must be diluted 5 orders of magnitude to reach the range of 30-300 colonies per plate. This dilution series adds inaccuracy to the assay. In the VCC procedure, by contrast, cells are not pipetted after the exposure to antimicrobial agents. Instead, twice-concentrated Mueller-Hinton broth is added and the 96-well plate is read for 12 hours in a plate reader. The VCC assay is much more precise. Its published results since 2005 in journals such as Antimicrobial Agents and Chemotherapy and The Journal of Biological Chemistry have demonstrated that the assay is highly reproducible except for paradoxical points, as is once again demonstrated in the present work for the triplicate results of HNP1+RNase at the high inoculum, assuming both preparations of RNase are equivalent. I encourage you to read the Ericksen 2020 reference, which includes a review of VCC studies up to that point and provides a peer-reviewed precedent for the use of VCC at high inocula. Sincerely, Bryan Ericksen, Ph.D."
},
{
"c_id": "8846",
"date": "24 Oct 2022",
"name": "William Jacobs",
"role": "Reviewer Response",
"response": "Dear Bryan, Thank you for your thoughtful response. Our lab has done death curves on Mtb for the last 10 years to look at the presence of persisters when subjected to bacteriocidal antibiotics. Typically we see a biphasic death curve that either leads to sterilization or drug resistance. I am not understanding with your technique how you are distinguishing amongst the four population: 1. live cells from, 2. dead cells, 3. persisters, and 4. drug resistant cells. While I await your answer, I will review the paper you cited. Furthermore, I did a search on Pubmed for the paper and cannot find it, can you provide the PMID number. Best, Bill"
},
{
"c_id": "9064",
"date": "13 Dec 2022",
"name": "Bryan Ericksen",
"role": "Author Response",
"response": "Dear Dr. Jacobs, Thank you for your detailed comments.  I will respond to each paragraph point by point. Initial paragraph: I am not sure what caveats you are referring to, but it is true that the VCC assay is sensitive not only to bactericidal killing but also to lag times.  Therefore, it can be regarded as an assay for general antibacterial activity, not killing, as is reflected in the title of the first VCC publication in 2005, âAntibacterial activity and specificity of the six human alpha defensinsâ.  It should be emphasized that the ability to cause bacterial cells to lag before growing could allow the rest of the innate and adaptive immune systems time to respond to the infection in vivo; therefore, both types of activity are significant.  VCC is an appropriate method for an initial study determining antibacterial activity, which can later be followed up by a study using the colony count assay determining bactericidal killing if a distinction is important between killing and causing lag times.  The advantages of VCC, such as using less media and the absence of a dilution series after exposure to the antimicrobial agent, make it ideal for a first study.  I feel the Ericksen 2020 reference adequately discusses all of these points and they do not need to be repeated in this publication.  As an initial study using only one method, I feel it is appropriate that this manuscript is a Brief Report, not a full-length article where one might see the results of multiple assay methodologies.  I would welcome a future publication by other researchers reporting colony forming units.  You mischaracterized my hypothesis somewhat.  I had to choose a convenient source of RNA for the study, so I chose tRNA, but my hypothesis is that any RNA, especially RNA normally secreted as part of bacterial growth or incorporated into biofilms, could inhibit antimicrobial peptides.  I do not mean to imply that tRNA is actively secreted by bacterial cells in a manner similar to other RNA, although undoubtedly some tRNA is released upon exposure to antimicrobial peptides due to the lysis of cells.  To emphasize this distinction, I have added âquite possibly a general property of RNA, not specific to tRNAâ to the sentence where the hypothesis is first described in the introduction. In addition, in the conclusions section I have added, âAny RNA, especially RNAs normally secreted as part of bacterial growth or incorporated into biofilms, could inhibit antimicrobial peptides.â  Your alternative hypothesis is certainly also occurring simultaneously: persister cells that do not grow are not killed by defensins, as was demonstrated in the 1980s by the original developer of the VCC assay, Robert I. Lehrer.  I consider a study of the phenomenon of persistence to be beyond the scope if this Brief Report.  Please note that I wanted to acknowledge Dr. Lehrer for sharing with me an unpublished manuscript hypothesizing that RNA inhibits defensins, but he has retired and did not respond to my request for permission to name him in the Acknowledgments section. Minor Comments: 1.   I am not sure what you mean by âdescribe all three togetherâ.  Do you mean include the results from all three in one long sentence?  I believe the abstract is ideal as is, because results are presented in the same order as in the figures.  I especially think it is appropriate to go through all the results for HNP1 first, since HNP1 was the primary focus of this study, with more emphasis and more experiments than LL-37 or HBD1. 2.   The words, âwith anâ were added to the methods section. 3.   You are correct that one of the Microsoft Excel shapes is a plus sign.  The legend shows both the shape and the line; therefore, the horizontal part of the plus sign cannot be distinguished from the line.  I was consistent in that I used âstandardâ and âhighâ to describe the inocula in the figure titles but used the CFU/mL in the legend keys.  Having both types of description in the same figure gives all relevant information succinctly.  I apologize for leaving the CFU/mL off of the legends for Figure 1 and Figure 6.  Unfortunately, the hard drive of the computer containing the Excel files failed, so these figures cannot be remade short of reading each value off the plots and manually reentering the data.  There is a long history of using this style of figure to present VCC results, as you can appreciate if you look at the seven Journal of Biological Chemistry publications cited in the Ericksen 2020 reference, for example. 4.   The title of Figure 3 has been edited to include âand HBD1 was assayed with tRNA.â  I apologize for the mistreatment of HBD1 in this study.  HBD1 was not assayed without tRNA in the experiment shown in Figure 3, nor was it assayed without RNase in the experiment shown in Figure 4, due to limited space available on the 96-well plate.  As such, the HBD1 data can be regarded as preliminary.  Further experiments using HBD1 would be of interest.  I considered omitting HBD1 from this manuscript but I decided to include it for completeness.  To improve the information presented on HBD1, I included âand HBD1+tRNAâ in the Abstract sentence mentioning cell clumps and in the Results section regarding cell clumps. 5.   Yes I expect this phenomenon is not limited to E. coli.  I look forward to future publications by other researchers testing other strains. 6.   Because data were not shown, the mention of washed cells has been deleted.  Although statistics cannot be reported for the single experiments using LL-37, a standard deviation can be calculated for the triplicate experiments using HNP1 shown in Figures 2, 5, and 6, assuming the two RNase preparations are equivalent.  The following was appended to the Results section: âThe results shown in Figures 2, 5, and 6 can be regarded as triplicate experiments, assuming the two RNase preparations are equivalent.  The mean virtual survival of HNP1 + 5x RNase was 0.75, and the standard deviation was 0.21.  The mean virtual survival of HNP1 + 25x RNase was 0.70, and the standard deviation was 0.10.  The mean virtual survival of HNP1 alone was 0.88, and the standard deviation was 0.02.  Based on these values, the two-tailed p-value for HNP1 + 5x RNase compared to HNP1 alone was 0.38, whereas the two-tailed p-value for HNP1 + 25x RNase compared to HNP1 alone was 0.10.  Therefore, the slight differences in activity observed were not statistically significant (p>0.05).â  Finally, the following was added to the Conclusions section: âAlthough the effect of HNP1+RNase was not statistically significant compared to HNP1 alone, it is possible that the effect of an antimicrobial peptide+DNase+RNase would be significant compared to the antimicrobial peptide alone.â 7.   I prefer not to speculate too much regarding the mechanism in the main text of the publication, but there are probably two mechanisms at work here: direct binding of the anionic RNA to the cationic antimicrobial peptide, and indirect induction of biofilm by RNA, in which case it is the biofilm that inhibits the antimicrobial peptide, not RNA itself.  It is possible that both mechanisms are important for HNP1, but only the indirect mechanism is important for LL-37.  This explanation highlights the assumption that hydrophobic interactions must be important for RNA binding to antimicrobial peptides, since electrostatic charge cannot explain this difference. 8.   I agree that a rigorous genetic analysis would be of interest and I hope the publication of this initial Brief Report encourages another researcher to undertake and publish those studies. 9.   I agree that the observed differences are slight.  I included the word âslightâ in the changes made in response to comment number 6, above.  A comparison with non-CAP antibiotics is beyond the scope of this study, but I would speculate that a 30% difference might be important simply because achieving any activity against biofilms at the high inoculum could be the difference between a lethal infection and an infection delayed such that the host immune system can mount an effective defense.  Any activity against biofilms might be important, especially given the minimal activity of HNP1 at the high inoculum."
},
{
"c_id": "9065",
"date": "13 Dec 2022",
"name": "Bryan Ericksen",
"role": "Author Response",
"response": "Dear Dr. Jacobs, The Ericksen 2020 reference is not indexed in Pubmed. It can be found in the peer-reviewed journal WikiJournal of Science: WikiJournal of Science/Virtual colony count - Wikiversity Dr. Lehrer and I speculated in the initial VCC publication (Antibacterial Activity and Specificity of the Six Human α-Defensins | Antimicrobial Agents and Chemotherapy (asm.org) that biphasic survival curves were due to the presence of persister cells. They may also explain paradoxical data, some of which is presented in this publication. However, optical density measurements in a plate reader cannot distinguish between actively growing cells and persisters. The study of persisters is well beyond the scope of this Brief Report, which is intended as an initial study using VCC alone."
}
]
},
{
"id": "155649",
"date": "18 Nov 2022",
"name": "Bouke K. H. L. Boekema",
"expertise": [
"Reviewer Expertise Microbiology",
"wound infection",
"antimicrobial peptides/therapies."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe idea of enhancing antimicrobial activity (of AMPs) is appealing and might be extended to other molecules/antimicrobials. Although there is a clear effect of tRNA on bacterial survival, the current paper is lacking in depth and does not meet the standards of scientific reporting. The introduction, materials & methods and results sections are rather limited. By what mechanisms might tRNA and RNase influence the AMP activities? There are several options which need to be addressed and experimentally tested.\nThe low number of AMPs approved for clinical use is mainly due to i) unfavorable pharmacokinetic profile, ii) safety issues and iii) reduced activity in clinically relevant environments. In view of this, the limited activity of the AMP-RNase combination does not show high potential.\nMinor Issues\nWhen treating more bacteria, it seems only logical that more antibacterial is required. Is it  biologically relevant if there is only bactericidal activity in case of low level of bacteria?\nAn alternative for VCC would be the use of a microcalorimeter for real time measurements.\nThe bacterial species is not mentioned in MM.\nWhy was the percentage TSB varied? And how would TSB affect activity?\nThe ratio tRNA to HNP, is this in mol/mol?\nAddition of RNase 25:1 to HNP1 enhanced activity (figure 2) but only at the highest concentration of HNP1.\nâLL-37 was enhancedâ (Figure 4)? Meant is that LL37 activity was enhanced? But in fig 4 more LL37 is needed for same effect as in fig3 (so it is not enhanced)? Please check/explain.\nâRNase was not active in the absence AMPâ: this is probably not true. RNase will still be active (against RNA) but not detectable as antimicrobial.\nFor all Figures: how many replicates were used, where is standard deviation/statistics?\nFigure 3: where are the results with HBD1 without tRNA?\nThere is mention of using washed cells but it is not clear how this was done (timing of washing).\nThe conditions for biofilm formation seem rather random, please elaborate.\nâpossible that tRNA inducing biofilmâ: is there evidence for this?\nFurthermore, I fully agree with the comments by reviewer Jacobs.\nIn case bacteria secrete peptide inactivating substances this can be tested by using culture supernatants.\nRestructuring the graphs would improve presentation of the results.\nThere is a clear effect of tRNA on bacterial killing, which might be demonstrated more clearly by using for example LIVE/DEAD stain, which does not involve dilution and culture and can be used on both low and high bacterial concentrations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9066",
"date": "13 Dec 2022",
"name": "Bryan Ericksen",
"role": "Author Response",
"response": "Dear Dr. Boekema, Thank you for your detailed comments. I will respond to each paragraph point by point in the same order as in your reviewer report. First paragraph: Although this article is lacking in depth and does not include mechanistic experiments, it is merely a Brief Report, intended as an initial study of antimicrobial activity only. Although the Introduction, Methods, and Results sections are limited, version 1 was very close to the 2500 word limit for a Brief Report. I cannot expand these sections without going over the word limit. I feel a Brief Report is appropriate because the results of only one method, VCC, are presented. Second paragraph: Although the activity of antimicrobial peptides in combination with RNase is slight, I feel it is significant because any activity at the high inoculum could be the difference between a lethal infection and an infection that the immune system has time to control in vivo, especially given the lack of activity of HNP1 alone at the high inoculum. Furthermore, these results are a stepping stone to antimicrobial peptide+DNase+RNase combinations, which may have statistically significant activity. As an initial study of an interesting phenomenon these results deserve to be published. Minor issues: Further studies are required to determine whether the amount of activity reported here is biologically relevant. I added the words, âare biologically relevant orâ to the first sentence in the last paragraph of the Conclusions section. I agree microcalorimetry can be used to detect antibacterial activity. I would look forward to a report by another researcher using such a method to confirm these results. âOf E. coli ATCC 25922â was added to the Methods section. The percentage of TSB was varied because it has two opposite effects. The nutrients increase activity, whereas the added salt decreases activity. I had been using 1% TSB for many years as instructed by Robert I. Lehrer, the original developer of the VCC assay, but I never tested other TSB concentrations until this article. I feel the results of the TSB variation are of interest to the study of defensins and so I included them in this article. The ratio of tRNA to HNP1 is wt/wt, not molar. I added âwt/wtâ to the Abstract. I added âat the highest tested concentration of HNP1â to the Abstract. The difference between Figure 3 and Figure 4 is that Figure 3 is at the standard inoculum, whereas Figure 4 is at the high inoculum. Therefore, Figure 4 does show an enhancement of activity due to the addition of RNase at the high inoculum. I added the word âactivityâ before enhanced in the Results section. I replaced the word âactiveâ with âantimicrobialâ regarding RNase alone. Figures 2, 5 and 6 can be regarded as triplicate experiments, as I mentioned in the response to Reviewer 1. Standard deviations and p-values have been added. The results of HBD1 without RNA were not tested because there was insufficient room on the 96-well plate. I apologize for the mistreatment of HBD1 in this article. In general, the emphasis of the article is on HNP1 more than LL-37, and LL-37 more than HBD1. Further studies of HBD1 are warranted. I considered omitting the HBD1 results but I included them for completeness. Because data are not shown, the mention of washed cells has been deleted. Biofilms formed at high inocula under the standard conditions of VCC assays. See the Ericksen 2020 reference for a further discussion. I added âsimilar to those previously described (Ericksen 2020)â to the Results section. The evidence that tRNA induces biofilm is the observation of cell clumps in the 96 well plate after the assay. I believe this evidence is clear from the article as is. I have fully responded to the comments of Reviewer 1. I agree testing culture supernatants would be interesting. I look forward to a future article presenting such results. There is a long history of this style of graph in VCC articles. For example, see the seven Journal of Biological Chemistry articles cited in the Ericksen 2020 reference. In any case, the hard drive of the computer containing the Excel files failed, so these figures cannot be remade. I agree that further experiments using LIVE/DEAD stain in conjunction with flow cytometry would be interesting, although well beyond the scope of this article, which is intended as an initial study using just the VCC method."
}
]
}
] | 1
|
https://f1000research.com/articles/11-933
|
https://f1000research.com/articles/12-267/v1
|
13 Mar 23
|
{
"type": "Research Article",
"title": "Seasonal effects decouple SARS-CoV-2 haplotypes worldwide",
"authors": [
"Tre Tomaszewski",
"Muhammad Asif Ali",
"Kelsey Caetano-Anollés",
"Gustavo Caetano-Anollés",
"Tre Tomaszewski",
"Muhammad Asif Ali",
"Kelsey Caetano-Anollés"
],
"abstract": "Background: Variants of concern (VOCs) have been replacing each other during the still rampant COVID-19 pandemic. As a result, SARS-CoV-2 populations have evolved increasingly intricate constellations of mutations that often enhance transmissibility, disease severity, and other epidemiological characteristics. The origin and evolution of these constellations remain puzzling. Methods: Here we study the evolution of VOCs at the proteome level by analyzing about 12 million genomic sequences retrieved from GISAID on July 23, 2022. A total 183,276 mutations were identified and filtered with a relevancy heuristic. The prevalence of haplotypes and free-standing mutations was then tracked monthly in various latitude corridors of the world. Results: A chronology of 22 haplotypes defined three phases driven by protein flexibility-rigidity, environmental sensing, and immune escape. A network of haplotypes illustrated the recruitment and coalescence of mutations into major VOC constellations and seasonal effects of decoupling and loss. Protein interaction networks mediated by haplotypes predicted communications impacting the structure and function of proteins, showing the increasingly central role of molecular interactions involving the spike (S), nucleocapsid (N), and membrane (M) proteins. Haplotype markers either affected fusogenic regions while spreading along the sequence of the S-protein or clustered around binding domains. Modeling of protein structure with AlphaFold2 showed that VOC Omicron and one of its haplotypes were major contributors to the distortion of the M-protein endodomain, which behaves as a receptor of other structural proteins during virion assembly. Remarkably, VOC constellations acted cooperatively to balance the more extreme effects of individual haplotypes. Conclusions: Our study uncovers seasonal patterns of emergence and diversification occurring amid a highly dynamic evolutionary landscape of bursts and waves. The mapping of genetically-linked mutations to structures that sense environmental change with powerful ab initio modeling tools demonstrates the potential of deep-learning for COVID-19 predictive intelligence and therapeutic intervention.",
"keywords": [
"AlphaFold2",
"epidemic calendar",
"membrane protein",
"mutation",
"protein interaction",
"protein structural prediction",
"proteome",
"seasonality",
"spike protein"
],
"content": "Introduction\n\nThe COVID-19 pandemic continues unabated. There have been more than 750 million total cases and 7 million total deaths worldwide, with about half a million new cases being reported every day (John Hopkins Coronavirus Resource Center). The makeup of SARS-CoV-2, the coronavirus responsible for the disease, is also changing at fast pace with the rise of numerous variants, some of which have impacted the success of vaccination and testing programs (Williams & Burgers, 2021; Talenti et al., 2022; McLean et al., 2022). Genomic changes arise from copying errors occurring during viral replication, the effects of host-induced editing (e.g. via host RNA deaminases), and recombination. Changes occur despite the SARS-CoV-2 genome being considered highly stable among positive-strand RNA viruses. This stability is endowed by its NSP14-mediated 3â-5â exoribonuclease proofreading activities, which repair polymerase errors (Ogando et al., 2020) but also mediate extensive viral recombination (Gribble et al., 2021). Within the context of evolving viral populations, the fate of mutations often depends on fitness (e.g. natural selection) or sampling (e.g. genetic drift, founder effects), by for example competitively enhancing viral replication, transmission rates, immune escape, or virulence. While too many deleterious mutations can push viral populations close to an âerror thresholdâ and the catastrophic possibility of extinction (Domingo et al., 2012), mutations that are not advantageous are generally eliminated. Instead, beneficial mutations often combine with each other to collectively increase the fitness of the viral âquasispeciesâ (Domingo et al., 1978), structuring this dynamic âcloudâ of viral variants by linkage and collective effects on function and fitness (Caetano-Anollés et al., 2022).\n\nEpidemiologically, viruses harboring either one mutation or unique constellations of mutations are generally referred to as âvariantsâ (Lauring & Hodcroft, 2021). These viral variants differ from âamino acid variantsâ describing mutations that cause amino acid substitutions, insertions or deletions. Mutations that are statistically or experimentally linked to clinical or epidemiological criteria of significance (e.g., virus transmissibility, disease severity, or immune or vaccine escape) are considered âmutations of concernâ (MOCs). Variants with MOCs become an immediate priority for surveillance and response. This is particularly so when their prevalence increases worldwide. In particular, variants exhibiting constellations of MOCs are considered âvariants of concernâ (VOCs). The first VOCs of SARS-CoV-2 appeared in October 2020, few months after the first wave of the pandemic. Since their appearance, circulating VOCs have been replacing each other, generally increasing the number of accumulating mutations in each replacement round. The World Health Organization has named these VOCs with Greek letters according to their times of origin and is planning to use names of celestial constellations thereafter.\n\nA constellation of mutations implies the existence of haplotypes (genetic signatures) and sets of mutations (markers, polymorphisms) that tend to be inherited together. Viral haplotypes represent mutations that are linked to each other, namely, that usually appear together in successful viral variants. This often results from beneficial intra-molecular or inter-molecular interactions operating at protein level. For example, the D614G mutation of the SARS-CoV-2 spike protein (S-protein), a substitution of an aspartic acid (D) by a glycine (G) at amino acid position 614 in the sequence of the S-protein, is part of a haplotype of four mutations including a P323L mutation in the NSP12 polymerase, a silent (F106F) mutation in the NSP3 papain-like protein, and a mutation in the 5â untranslated region (UTR) of the genome. This first gene set was fixed in the worldwide viral population during the first wave (April 2020) of the COVID-19 pandemic (Tomaszewski et al., 2020). The haplotype is believed to have increased infectivity by enhancing the flexibility of the S-protein (Voltz et al., 2021). The S-protein is a trimer of highly glycosylated protomers, each harboring a N-terminal S1 subunit sequence with an N-terminal domain (NTD) and a receptor-binding domain (RBD), and a C-terminal S2 subunit holding a âfusionâ region and a âtransmembraneâ region. The D614G mutation breaks a D614-T859 side chain hydrogen bond between the neighboring S1 and S2 subunits of pairs of protomers enhancing flexibility and subunit interactions (Korber et al., 2020). Cryo-electron microscopy (cryo-EM) analysis revealed that the mutation disrupted interprotomer contacts shifting the conformation towards an ACE-2-binding competent state necessary for membrane fusion with target cells (Yurkovetskiy et al., 2020). A subsequent conformational dynamics study showed that the mutation also affects the residues K854 and Y837 of the fusion peptide (FP) region contributing to linkage and/or allostery between the subunits (Xu et al., 2021). All VOCs have retained this initial haplotype but have added others as they appeared and were replaced, with each new constellation gathering a larger and more durable haplotype set by haplotype coalescence.\n\nSince major VOCs such as Delta and Omicron have overtaken the entire global viral population, there is an implicit assumption that VOC mutant constellations represent relatively stable haplotypes. We recently tested this assumption by exploring the appearance and accumulation of VOC constellations in Australia as these were emerging throughout the COVID-19 pandemic (Tomaszewski et al., 2022). We chose Australia for two reasons. First, the country was able to control infection for the majority of the pandemic through effective disease mitigation strategies, including closing borders of states and country. This provided grounds to explore haplotype emergence in absence of significant effects from host migrations. Second, the Australian population is sparsely distributed and clumped into clearly defined urban areas along a significant latitude transect. This allowed testing effects of seasonal behavior on patterns of mutation accumulation since the beginning of the pandemic. The study revealed that 20 major haplotypes were associated with VOCs Alpha, Delta and Omicron in Australia and that there were significant recruitment episodes. Remarkably, core constellations showed significant decoupling patterns suggesting processes of emergence and significant and unanticipated seasonal patterns of diversification were at play in Australia. Decoupling manifested as latitude-imposed idiosyncratic patterns of accumulation within and between haplotypes.\n\nHere we extend our exploration of haplotypes to the entire world. The GISAID initiative, sponsored by governments in partnership with public health and research institutions, has created a repository of genome data collected by extensive worldwide genome sequencing efforts (Elbe & Buckland-Merrett, 2017; Shu & McCauley, 2017; Khare et al., 2021). We mined levels of genetic variation unfolding in the evolving viral population using over 12 million genomic sequences retrieved from the GISAID repository on July 23, 2022. The construction of an âhaplotype networkâ that describes the worldwide viral population landscape throughout the COVID-19 pandemic confirmed significant decoupling patterns and increasing coalescence of haplotypes into larger haplotype groups. We also revealed seasonal patterns of emergence and diversification amid a highly dynamic viral evolutionary landscape. A protein interaction network mediated by haplotypes predicted molecular interactions, the effects of which could be tested with powerful ab initio structural modeling tools at atomic resolution. These results provide a unique window into our evolutionary understanding of a human pathogen of great significance.\n\n\nMethods\n\nThe metadata for 12,070,698 SARS-CoV-2 genome sequences were downloaded on July 23, 2022, from the GISAID EpiCoV⢠repository. Importantly, the metadata for each sequence contained a field listing all amino acid substitutions, referenced against the âhCoV-19/Wuhan/WIV04/2019â sequence (GISAID accession ID: EPI_ISL_402124, GenBank accession version: MN908947.3) (Wu et al., 2019). After extraction, data were filtered, limiting inclusion to sequences that were (a) obtained from a human host and (b) either labeled as âcompleteâ or âhigh-coverageâ. After filtering, metadata for 11,923,363 sequences remained, and these fields, along with other task-unnecessary metadata, were removed. See supplementary acknowledgements for the complete list of Accession IDs used.\n\nTo compare the prevalence of substitutions in climate zones, we split the âlocationâ metadata field into the component parts of continent, country, region, and sub-region. The resulting country names were normalized for consistency and manually mapped to match a list of country coordinates assembled from a canonical dataset (available from DSPL, Google). The latitude was added as a field in the sequence metadata. These were then used to label a climate zone for each sequence. The scheme used five climate-zones: 30°S to 30°N latitudes as âTropicsâ, 30°N to 60°N latitudes as âNorthern Temperateâ, 30°S to 60°S latitudes as âSouthern Temperateâ, and 60°N to 90°N latitudes as âArcticâ. No âAntarcticâ sequences corresponding to 60°S to 90°S latitudes were available. To identify the temporal dimension, we used monthly enumerations listing the month and year of sequence collection dates, starting on December 2019 (as index #0) and ending on July 23, 2022 (index #31).\n\nAmino acid substitutions were isolated by collecting all variations occurring at least once within the lists present in the metadata âAA substitutionâ field for each sequence. They were labeled using accepted nomenclature (den Dunnen & Antonarakis, 2000) from the Human Genome Variation Society. After aggregation, each of the 183,276 unique substitutions was split into component parts: (a) the protein name, (b) the reference amino acid, (c) the location of the amino acid in the protein, and (d) the amino acid substitution (deletion or insertion). The number of sequences per substitution over the entire period from December 2019 to July 2022 was counted. Since substitution groups containing low variance or spurious accumulation patterns were undesired for further analysis, they were filtered using a ârelevancyâ heuristic. We analyzed only substitutions with sequence counts greater or equal to one standard deviation over the mean of the entire sequence set.\n\nEach sequence was indexed by the GISAID accession ID, climate zone, and monthly index, then (multi-)classified by amino acid substitution using a one-hot encoding (i.e., â1â identified the existence of the substitution in a sequence and â0â an absence). The incidence of each amino acid substitution for each climate-zone and month grouping was then calculated by summation across sequences and then divided by the total number of sequences within the climate zone-temporal period. For visualization, the monthly incidence of each climate zone was depicted in individual plots for each unique substitution using the Python library âmatplotlibâ v. 3.5.2 (Caswell et al., 2021; Hunter, 2007) and arranged and annotated using Adobe Illustrator v.25.2.3.\n\nAccelerated ab initio modeling of 3-dimensional atomic structures was conducted using the AlphaFold2 pipeline (Jumper et al., 2021) implemented locally in ColabFold without changes or modifications (Mirdita et al., 2022). The output of five ranked structural models was obtained following three neural network recycles (processing of predictions through models) that iteratively extracted co-evolutionary information in PDB70 structural templates and multiple sequence alignments (MSAs) for end-to-end training of the deep learning âevoformerâ and âstructureâ multi-layered neural network modules. MSAs were built with fast and sensitive MMseqs2-based homology searches of UniRef100 and a database of environmental sequences. Accuracy was measured with the predicted local distance difference test (pLDDT) and the predicted aligned error (PAE). pLDDT provides a per-residue estimate of prediction confidence based on the LDDT-Cα metric (Mariani et al., 2013). The expected prediction reliability of a given region or molecule follows pLDDT âconfidence bandsâ: >90, models with very high confidence; 90-70, models with confidence, showing good backbone predictions; 70-50, models with low confidence; and <50, models with very low confidence, generally showing ribbon-like structures. pLDDT <60 can be considered a reasonably strong predictor of intrinsic disorder. PAE measures confidence in the relative positions of pairs of residues, which evaluates the cohesiveness of structural modules (e.g., domains). Structural alignments and visualizations were carried out using Chimera (Pettersen et al., 2004). Reference (corresponding to M-EPI_ISL_402124) and variant structures were superimposed using the MatchMaker and MatchAlign tools to identify regions with structural divergences. Topological similarities of individual regions or entire molecules were evaluated with average template modeling scores (TM-scores; Zhang & Skolnick, 2004) using USalign (Zhang et al., 2022a). M-protein predictions were benchmarked against two structural conformations recently acquired using cryo-EM (Zhang et al., 2022b). Besides TM-scores, GDT-TS scores were obtained using the LGA (local-global alignment) structure comparative analysis tool (Zemla, 2003) with the AS2TS server, which CASP assessors routinely use to evaluate the accuracy of predicted structural models.\n\n\nResults\n\nSARS-CoV-2 variants are organized around a master genomic sequence representing the virus that originated in the city of Wuhan in China. Many mutations have been added (some subtracted) to this master sequence since the beginning of the pandemic. These genomic changes can be traced by conducting a phylogenomic analysis of genomic sequences sampled throughout the disease timeline. Figure 1A shows a timetree reconstructed using the Nextstrain pipeline. It represents one out of 1,000 built from randomly sampled taxa. The trees have an implied time axis (measured in months) that allows dissecting viral spread, variant introductions, and rates of genomic change. Splits in the branches of the trees recovered using maximum likelihood optimality criteria define clades, groups of taxa with a common evolutionary origin (colored circles in the figure). They also define hypothetical ancestors embodying vectors of genomic sites that differ from the master sequence (the ancestor of all taxa) and change along branches as shared and derived phylogenetic features. These ancestors were recovered using character state reconstruction methods. As of July 2022, 26 Nextstrain clades defined by a year-letter nomenclature (beginning with clade 19A) and differing by at least two mutations from their parent major clades (Hodcroft et al., 2020) cataloged global viral diversity. Some splits in the tree describe the evolutionary appearance of major lineages. Figure 1B also tracks the accumulation of genomes corresponding to different VOCs, revealing how VOCs and their associated clades are being continuously replaced. For example, the VOC Omicron wave was represented by the clades 21K, 21L, 22A, 22B and 22C, which originated from a larger, more basal clade that at the very beginning of the pandemic gave rise to lineages that later led to VOC Alpha. Only three VOCs, Alpha, Delta and Omicron, became predominant worldwide at some point, completely replacing each other. VOC Alpha (clade 20I) appeared in the United Kingdom and was the first to expand quickly and globally probably due to increases in transmissibility and infection rates (Davies et al., 2021). VOC Delta (mostly 21J) was first identified in India in October 2020 but became predominant worldwide in June 2021. Finally, VOC Omicron was discovered in Botswana and South Africa in November 2021 before quickly spreading throughout the world in 2022.\n\n(A). A maximum likelihood phylogenetic tree describing the worldwide history of the SARS-CoV-2 genome. The timetree of 2,906 genomes randomly sampled between December 2019 and July 29, 2022 was obtained from Nextstrain. The tree unfolds the time of genome collection date from left to right. Its leaves (taxa indicated with circles) are colored according to the clade (group of taxa with a common evolutionary origin) and emerging variants of concern (VOCs) nomenclature. The origin of VOCs occurs when a clade originates along branches of the phylogeny. Note the early arrival of VOC Alpha, followed by VOC Delta and then VOC Omicron. The timeline of clades and VOCs shows three successive phases driven by proteome flexibility and rigidity, environmental sensing, and vaccine-driven immune escape, which are shaded in light yellow, blue, and salmon, respectively (Caetano-Anollés et al., 2022). (B). Plots showing the number of daily newly confirmed cases per million people (on a logarithmic scale and as 7-day rolling averages) and smooth percentages of genomes holding major VOCs since the beginning of the recorded COVID-19 pandemic. COVID-19 and genome data were retrieved from Johns Hopkins Univ., CSSE and GISAID, respectively. (C). Spike map showing a 3-dimensional representation of the population density of the world as a grid of vertical bars depicting the number of people per square kilometer of land area. Each spike represents the population in a grid of 2 km à 2 km. Light and shadow effects on the map highlight areas of high population density but also isolated population centers. Note the map shows no land. Instead it highlights locations where the 7.8 billion people of the world live (as of 2020). Labeled latitudes were used to split the world into four regions: Arctic, Northern Temperate, Tropics, and Southern Temperate, which are identified with colored letterings on the map and used to divide the genomic pool of the virus. The spike map is courtesy of Alasdair Rae, Automatic Knowledge Ltd., Sheffield, UK, reproduced with permission.\n\nTo track the prevalence of individual mutations emerging throughout the pandemic, we analyzed 11,923,363 genomic sequences drawn from four climatic regions delimited by latitude worldwide (Figure 1C), partitioning them into those acquired in each month of the pandemic. Regions included âTropicsâ, âNorthern Temperateâ, âSouthern Temperateâ, and âArcticâ (latitude boundaries defined by 30°, 60° and 90° N and S); no âAntarcticâ sequences were available. Given background knowledge, latitude-delimited regions were expected to uncover seasonal effects in the global viral population (Caetano-Anollés et al., 2022). From these sequences, a total of 183,276 unique amino acid substitutions and insertion/deletions (variants) were identified and subsequently filtered with a ârelevancyâ heuristic that only kept those with a prevalence of greater or equal to one standard deviation over the mean of the entire sequence set. Note that the threshold, months, and regions are dynamic, and that the filtering criterion guarantees that we are not missing any significant mutations, especially those considered VOC markers. Accumulation plots of individual mutations corresponding to the major VOCs Alpha, Delta and Omicron (Figure 2) were generated from the data and accumulation given as a prevalence. Prevalence ranged from 0 to 1, with 1 implying that 100% of genome sequences collected during an individual month contained that mutation. Collectively, accumulation plots describe the set of the most significant mutations affecting individual proteins of the viral proteomes in the different regions of the world and during each month of the pandemic.\n\n(A) Accumulation plots illustrating haplotypes emerging along a timeline of the pandemic with labels colored according to VOCs they belong and time unfolding from top to bottom. The accumulation plot of a single mutant illustrates each haplotype. (B) Accumulation plot overlaps of all mutant markers of haplotypes describe haplotype decoupling for individual climatic zones. (C) Accumulation plots for mutants belonging to each haplotype are displayed from left to right. Mutant names are colored according to the VOCs they belong. The inset shows accumulation plots for free-standing markers.\n\nAccumulation plots allowed us to define individual haplotypes, study their rise along a timeline, and evaluate their cohesiveness in the different climate zones of the world (Figure 2). We defined haplotypes as sets of mutations harboring the same or very similar accumulation patterns during the lifetime of a VOC constellation (Figure 1A). The cohesiveness of molecular and physiological interactions of a haplotype is however diminished when one or more mutations of a haplotype show distinct accumulation patterns. This âdecouplingâ property can be uncovered by overlapping mutation accumulation plots for the four climate zones (Figure 2B). Mutations were indexed according to their presence in the three widespread VOCs of the pandemic, Alpha, Delta and Omicron (Figure 2C). We identified 22 haplotypes composed of 2â18 mutations affecting 1-8 proteins, one shared by the three VOCs and three shared by VOCs Alpha and Omicron. Two additional haplotypes belonged to minor VOCs listed in the accumulation plots of other markers identified with the relevancy heuristic (Figure 3). Five free-standing mutations also unified the VOCs Omicron and Delta. Markers of VOC constellations confirmed the recruitment patterns we previously uncovered in Australia (Tomaszewski et al., 2022). Haplotype constitution was relatively conserved but also showed it was subject to the increasing trend of marker and haplotype accretion we highlighted worldwide (Caetano-Anollés et al., 2022) and in Australia (Tomaszewski et al., 2022). Indeed, we found five haplotypes holding 23 markers for the early VOC Alpha, six haplotypes holding 28 markers for VOC Delta, and 16 haplotypes holding 78 markers for the ongoing VOC Omicron. Thus, haplotypes and their associated markers have increased over time during our genomic sampling period. We note that six out of the 22 haplotypes we identified did not attain significant prevalence during the pandemic period analyzed; all labeled with prime symbols. For consistency, we named haplotypes following the nomenclature used in our Australian genomic study (Tomaszewski et al., 2022).\n\nPrevalence plots describing the accumulation of minor markers that failed to achieve large prevalence levels but were retained by the relevance heuristic. Note the existence of two cryptic haplotypes associated with the rise of VOC Alpha in Tropical and Southern Temperate corridors (C1 and C2).\n\nThe first haplotypes appearing worldwide were haplotype 5 and haplotype 2, in that order. Haplotype 5 involves the well-known 4-mutation set mentioned above that includes the nonsynonymous D614G amino acid substitution of the S-protein and the P323L substitution of the NSP12 polymerase. The haplotype was first established in Europe during the first wave of the pandemic before spreading to other areas of the world. It is the most stable so far, is present in all VOCs, and is believed to increase COVID-19 infectivity (Becerra-Flores & Cardozo, 2020; Korber et al., 2020). Figure 2 shows that the emergence of the haplotype was noisy and decoupled across regions until November 2021, especially in the Arctic, Southern Temperate and Tropical regions of the world. Haplotype 2 involves two mutations located in the intrinsically disordered serine/arginine-rich linker that separates the N-terminal and C-terminal RNA-binding domains of the nucleocapsid protein (N-protein), R203K and G204R (Tomaszewski et al., 2020). Mutations were tightly coupled worldwide except for regions of the Northern Hemisphere during the first two quarters of 2021. Mutation prevalence was significant throughout the pandemic except for August-November 2021 (immediately before the rise of VOC Omicron), when mutation incidence decreased significantly worldwide. Prevalence patterns revealed two hemisphere-related waves, one centered around July 2020 that diminished prevalence in the Northern Temperate region, and another centered around April 2021 that fostered (decoupled) prevalence in Northern Temperate and Arctic regions. These patterns and the rise of the haplotype during the winter in the Southern Hemisphere suggest a seasonal effect. A similar conclusion was drawn from mutant accumulation plots in Australia (Tomaszewski et al., 2022). The haplotype was effectively recruited into VOC Omicron.\n\nThe mutant constellation of VOC Alpha introduced three additional haplotypes composed of 4-12 mutations affecting 1-4 proteins, haplotypes 4, 3, and 1, in that order (Figure 2). The three haplotypes had larger incidences in the Northern Hemisphere, especially the last two. Their recruitment coincided with the second wave of haplotype 2 we described above. Haplotype 4 first appeared in the Southern Temperate region as a tightly linked set of four mutations, the N501Y mutant of the S-protein and three mutations in the autophagy-associated NSP6 protein that is linked to SARS. The haplotype extended relatively quickly worldwide, was tightly linked to markers of haplotype 2 from July to November 2021 and was then coopted by VOC Omicron. Haplotype 3 involved three mutations (deletions) affecting the NTD of the S-protein. Their accumulation followed that of haplotype 4 but their incidence vanished from August to November 2021 before being coopted by VOC Omicron. Finally, haplotype 1 involved 12 mutations affecting the S-protein, N-protein, the accessory ORF8 immune evasion protein, and the NSP3 papain-like proteinase scaffold. Markers belonging to each of all three haplotypes were tightly linked with each other, a pattern that differs from those of subsequent VOCs. As expected, however, prevalence of the three haplotypes across regions was low (below about 10% to 90%) following the low worldwide prevalence of VOC Alpha (Figure 1B).\n\nThe mutant constellation of VOC Delta introduced five additional haplotypes harboring 2-11 mutations affecting 1-7 proteinsâthe S-protein, N-protein, membrane M-protein, ORF3, ORF7a, ORF7b, NSP3 protease, NSP4, NSP6, NSP12, and NSP14 exonuclease (Figure 2). The widened diversity of haplotype proteins suggests VOC Delta significantly enhanced inter-molecular interactions. Except for haplotypes 9â and 10â, haplotype prevalence reached 70% to 100% in all regions, with lower prevalence consistently evident in Tropical and Southern Temperate regions.\n\nFinally, the core mutant constellation of VOC Omicron introduced an additional 12 haplotypes containing 2-18 mutants affecting 1-8 proteinsâthe S-protein, N-protein, M-protein, envelope protein (E-protein), ORF3, NSP1, NSP3, NSP4, NSP5, NSP6, NSP12, NSP13 and NSP14 (Figure 2). Haplotypes 13, 17, 19, and 23â altered sites exclusively present in the S-protein. Haplotypes 12, 14, and 16 also involved a significant number of S-protein markers. Overall, 37 out of 54 markers (69%) in these 12 haplotypes altered the S-protein, contrasting with only 14 S-protein markers out of 49 markers (29%) present in preceding haplotypes. This showed that the mutational landscape of the virus was becoming significantly biased towards the S-protein as the pandemic unfolded. Accumulation plots reveal most VOC Omicron haplotypes gained 100% prevalence in 3-4 months since the detection of the VOC in the Gauteng Province of South Africa in November 2021, much quicker than the six months it took for other VOCs to reach global or maximum prevalence levels. Haplotypes 12 and 15 were the first to accumulate and reach solid 100% prevalence. Haplotypes 13, 18, and 19 followed the same accumulation pattern but failed to reach global prevalence in Tropics and Southern Temperate regions. In turn, haplotypes 14 and 17 struggled to reach 80% prevalence in January 2022 but then their incidence decreased in all regions, vanishing in Arctic and Southern Temperate regions. Conversely, Haplotype 16 increased at a lower rate after a lag of a month only to reach 100% prevalence in the Arctic and Southern Temperate regions. The late appearing minor haplotypes 20â-23â showed vanishing tendencies or late increasing trends. Overall decoupling patterns suggest significant impact of latitude-related effects on the evolving genomic makeup of VOC Omicron.\n\nOverlaps of accumulation plots for mutants in each haplotype revealed an increasingly significant decoupling of the VOCs Delta and Omicron haplotypes, with exceptions in haplotypes 6, 8, 10â, 12, 18, 20â, 21â, and 23â (Figure 2B) Decoupling regions were located in both the Northern and Southern Hemispheres. For example, haplotype 15 was particularly decoupled in the Southern Temperate region while haplotype 13 was particularly decoupled in the Arctic region.\n\nAccumulation plots show that several haplotype markers appeared earlier in the pandemic than the haplotypes themselves (Figure 2). As observed in Australia (Tomaszewski et al., 2022), the N-protein variant P13L of haplotype 15, which is associated with the N-terminal region of the nucleocapsid that is intrinsically disordered (Tomaszewski et al., 2020), appeared during the first wave of the pandemic between March and June of 2020 in the Tropics and Southern Hemisphere. The mutation was part of a pathway of mutational change involving protein flexibility/rigidity (Tomaszewski et al., 2020). The marker likely represents the oldest mutation of VOC Omicron other than those of haplotypes 2 and 5. Similarly, S-protein markers H655Y and P681H of haplotype 12 appeared during the rise of VOC Alpha and S-protein markers K417N and S477N of haplotype 19 appeared earlier in 2020. We also note that the A67V and V143del mutants of the S-protein and the D3G mutant of the M-protein of haplotype 14 appeared before VOC delta in 2020. All of these patterns support the existence of significant recruitment operating during haplotype emergence (Tomaszewski et al., 2021). This emergence is likely mediated by recombination, the process in which genomes of variants combine to form new variants during the replicative cycle of the virus.\n\nThe existence of a cloud of viral variants exploring a combinatorial landscape of mutations predicts that mutations and their combinations should precede the rise of VOC constellations. In a previous study we showed that VOC haplotypes recruited marker combinations already present in individual protein sequences before VOC emergence during late 2020 (Caetano-Anollés et al., 2022; Tomaszewski et al., 2022). Here, we illustrate again the reuse of marker combinations in haplotypes by studying their presence in a dataset of 137,605 sequences of the S-protein retrieved worldwide on November 14, 2020 by Showers et al. (2022), exactly one month earlier than the announcement of VOC Alpha in the United Kingdom (Public Health England, 2020). Counting the number of mutations in the S-protein sequences surveyed in the benchmarking study showed that most sequences harbored between 1-3 mutations with an average of 2.53 ± 0.94 (SE) mutations per sequence (Table 1). The survey revealed that out of the 2,942 variant combinations identified, there were only 13 combinations with six mutations, one each with seven and eight mutations, and two with nine mutations in the data set. One of the two nine-mutant combinations (H69_V70del-Y145del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H) was present in 22 sequences and contained all VOC Alpha S-protein markers, including those of haplotype 1 (A570D, T716I, S982A, D1118H), haplotype 3 (H69del, V70del, Y145del), haplotype 4 (N501Y) and haplotype 5 (D614G), and the free-standing marker P681H, which collectively characterize the S-protein constellation of this viral variant. Note that Y144del of haplotype 3 is indexed as Y145del in the dataset due to difficulties with identical adjacent amino acids in the alignment software (Showers et al., 2022). All 17 sequences contained at least one if not two to three of these markers, suggesting mutation increase in S-protein sequences was prerequisite for VOC emergence. Figure 4 maps the prevalence levels of the VOC Alpha mutations in the combinatorial landscape of 2020. The plot shows a strong bias in the prevalence of mutant combinations holding VOC Alpha markers, supporting a mechanism of VOC emergence via rearrangement rather than selective sweep in the viral population. Thus, going back to the first COVID-19 wave of 2020 reveals that VOC Alpha emerged by a combinatorial rearrangement of mutations already existent at different prevalence levels in the variant population.\n\n* Average: 2.53±0.94 (SE) out of 2,942 variants. Regional deletions were counted as one mutation.\n\nList of combinations containing six mutations with VOC Omicron haplotype markers labeled in bold and free-standing markers in italics:\n\nH69_V70del,L189F,N439K,D614G,V772I,G1219V (62 sequences)\n\nD80Y,N164T,A222V,A262S,D614G,P1140X (18 sequences)\n\nL5F,A222V,D574Y,D614G,H655Y,P1140X (11 sequences)\n\nD80Y,Y145del,N164T,A222V,A262S,D614G (5 sequences)\n\nD80Y,T95I,N164T,A222V,A262S,D614G (4 sequences)\n\nL5F, A222V,D574Y,D614G,H655Y,K1205N (4 sequences)\n\nD80Y,N164T,A222V,A262S,D614G,W1214X (3 sequences)\n\nD80Y,S98F,N164T, A222V,A262S,D614G (3 sequences)\n\nL5F, A222V,D574Y,D614G,H655Y,W1214X (3 sequences)\n\nL5F, H69_V70del,L189F,N439K,D614G,V772I (2 sequences)\n\nH69_V70del,L189F,N439K,D614G,A647V,V772I (2 sequences)\n\nL5F, A222V,N536S,D574Y,D614G,H655Y (2 sequences)\n\nD215V, A222V,D614G,P1140X,D1163Y,G1167V (2 sequences)\n\nList of combinations containing seven or more mutations with VOC Omicron haplotype markers labeled in bold and free-standing markers in italics:\n\nL141_V143del,Y144F,T478K,E484K,S494P,D614G,I870V(2 sequences)\n\nF65L,H66L,H66_A67insG,A67S,I68M,V70I,N439K,D614G (3 sequences)\n\nH69_V70del, Y145del,N501Y,A570D,D614G,P681H,T716I,S982A,D1118H (22 sequences)\n\nL141_V143del,Y144F,Q183H,T478K,Q493K,S494P,N501Y,D614G,I870V (2 sequences)\n\nThe plot is indexed with the names of 83 combinations harboring markers of the VOC Alpha constellation (in bold) and corresponding prevalence (number of sequences in parentheses). Note that VOC Alpha was reported a month after the sampling of the 137,605 S-protein sequences analyzed on November 14, 2020. Markers highlighted in blue have a higher prevalence than the 22 sequences of a single mutant combination harboring all markers of VOC Alpha (highlighted in green). They represent 67% of markers of that combination, offering ample opportunities for recombination. The inset shows a network of co-occurrence of markers of the VOC Alpha constellation. Nodes are mutations and links of the graph represent their co-occurrence. Data were retrieved from the Supplementary Tables in Showers et al. (2022).\n\nRemarkably, 58% of the VOC Omicron haplotype markers that appeared at the end of 2021 were already present in the accumulating 2,942 mutant combinations of November 2020, most of them at low prevalence levels (Table 2). Haplotypes 3, 4, 5, 13, 15, and 16 had markers present in the 17 mutant combinations that had 6-9 mutations. Except for haplotypes 15 and 22â, all VOC Omicron haplotypes had markers representing them. Thus, haplotype primordia were already forming in the evolving viral population.\n\nThe table lists the number of distinct mutant combinations harboring VOC Omicron markers out of the total 2,942 identified in a set of 137,605 sequences of the S-protein retrieved worldwide on November 14, 2020 (Showers et al., 2022).\n\nWe constructed a âhaplotype networkâ describing the haplotype and mutant makeup of major VOCs (Figure 5). The nodes of the graph are either haplotypes or free-standing mutations coalescing into VOC-specific mutant constellations. Node size is proportional to the number of haplotype markers. Edges describe common patterns of prevalence in accumulation plots. Circles portray levels of haplotype coalescence, that is, similarities in patterns of mutation accumulation of haplotype markers. Circles closer to the middle harbor mutants and haplotypes with prevalence patterns that are both similar and unique to each VOC constellation. Outer circles host mutants and haplotypes with patterns that are either more variable or shared by constellations of different VOCs. Mechanistically, haplotype coalescence unifies constellations pushing nodes to the middle of subgraphs and fostering hub behavior. In turn, seasonal decoupling, marker loss, and recruitment (sharing between constellations) push haplotypes and markers to their periphery. This frustrated interplay illustrates the dynamic mutational landscape of the virus.\n\nNodes and edges of the graph describe how haplotypes and free-standing mutations coalesce towards the inner-most circles of the major VOC constellations. Haplotype and mutant labels are colored according to their presence in VOCs worldwide. Cryptic markers are listed in Figure 3.\n\nThe network in Figure 5 shows three clear subgraphs corresponding to the VOCs Alpha, Delta and Omicron being unified by the universally present haplotype 5 and other shared haplotypes and free-standing markers. The network is structured by recruitment, coalescence, decoupling and loss:\n\n(i) Recruitment: The constellation of VOC Omicron recruited three haplotypes (haplotypes 2, 3 and 4) and one free-standing S-protein mutant (P681H) from VOC Alpha, and five free-standing mutants from VOC Delta. Together with haplotype 5, these recruitments impacted the S-protein (10 markers), N-protein (2 markers), NSP4 (1 marker), NSP6 (3 markers) and NSP12 (1 marker). Remarkably, VOC Alpha contributed almost half of its markers (11 out of 24 total) and 4 out of its 5 haplotypes to VOC Omicron, while VOC Delta contributed only a fifth of its markers (7 out of 35 total) and only 1 out of its 6 haplotypes to the makeup of the new VOC. Each episode of recruitment challenges the cohesive properties of the newly formed constellation because recruited haplotypes carry their own accumulation idiosyncrasies. For example, markers of haplotype 5 were poorly linked during the rise of VOC Alpha but highly linked during the rise of VOC Omicron (Figure 2). The reverse was true for markers of haplotypes 3 and 4.\n\n(ii) Coalescence: We identified an apparent correlation between the time of origin of VOCs and haplotype coalescence and size. The 24 markers of the VOC Alpha constellation coalesced into five haplotypes, about half of which formed a single haplotype (haplotype 1). Only one marker remained free-standing. The 35 markers of VOC Delta coalesced into six haplotypes, a third of which formed the largest haplotype (haplotype 7) of the set. Seven markers remained free-standing. In sharp contrast, the 92 markers of VOC Omicron organized into 16 haplotypes, about a fifth of which formed the largest haplotype (haplotype 16). 14 markers remained free-standing. The VOC Omicron constellation became stratified into a central core with at least three layers, one holding haplotypes 12 and 18, another holding haplotype 16, and a third holding haplotypes 13, 14, 15, 17, and 19. In addition, four peripheral haplotypes joined haplotypes recruited from VOC Alpha and a host of free-standing markers. Clearly, as VOCs replaced each other, markers increased in number but their constellations fragmented into increasingly more numerous haplotypes. The diversity of accumulation patterns also increased with the time of origin of VOC constellations. The typical single (sometimes noisy) single-peaked burst of mutation accumulation of VOC Alpha was replaced by multiple tightly overlapping bursts in VOC Delta and by both multiple-rate overlapping sigmoidal accumulations and distinct overlapping bursts in VOC Omicron (Figure 2).\n\n(iii) Decoupling: Markers tightly linked to each other in a haplotype are expected to show minimum differences in accumulation unless the molecular interactions or physiologies they mediate are affected by environmental, behavioral or physiological drivers. In our study, the decoupling effects of latitude on mutation prevalence tested the cohesiveness of individual haplotypes in VOC constellations. The monolithic behavior of haplotype 1 of VOC Alpha, haplotypes 6 and 8 of VOC Delta, and haplotypes 12 and 18 of VOC Omicron placed them at the core of their respective constellations, while more variable haplotypes were more peripheral (e.g. haplotypes 4, 7 and 16). Recall that decoupling manifests as idiosyncratic patterns of accumulation within and between haplotypes. VOC Alpha constellations hosted two types of haplotypes, one widely prevalent and exhibiting highly consistent accumulation patterns in the Northern Hemisphere (haplotypes 1 and 3), the other quite noisy. Similarly, the steady gain of the high prevalence of most haplotypes of VOC Omicron was countered by the âburstâ behavior of haplotypes 14 and 17 driven by the replacement of the initial 21K clade of VOC Omicron (Figure 1B).\n\n(iv) Loss: In contrast with the steady and highly prevalent levels of haplotype 5, the early but steady haplotypes 2, 3, and 4 showed episodes of gain and loss along the timeline of the pandemic (Figure 2). The haplotypes appearing later were lost once VOCs started replacing each other. Free-standing markers were also lost, except for G142D and T478K of the S-protein and T402I of NSP4, which increased with VOCs Delta and were recruited by VOC Omicron. Free-standing marker P681H shared by the VOCs Alpha and Omicron was lost in October-November 2021 but was then regained (Figure 2C).\n\nA global analysis of COVID-19 seasonal behavior during the early stages of the pandemic showed that effective disease transmission was restricted to a 30° to 60° latitude corridor in both the Northern and Southern Hemispheres (reviewed in Caetano-Anollés et al., 2022). An initial study however failed to reveal an association between genomic and epidemiology data (Burra et al., 2021), perhaps because the focus was global genomic change levels during the first wave of the pandemic. In contrast, overlaps of accumulation plots for mutants belonging to individual haplotypes already uncovered distinct latitude-dependent accumulation trends in our study (Figure 2B). These trends dissected the tropics from the temperate regions of the world. To better visualize these accumulation patterns we selected haplotypes that coalesced into the highly cohesive cores of the three haplotype subnetworks in Figure 5. We excluded haplotypes arising before the first appearance of VOCs (haplotypes 2 and 5) and those highly variable shared by the VOCs Alpha and Omicron, focusing instead on haplotypes with minimum decoupling. The haplotype core of VOC Alpha involved only haplotype 1, while those of VOC Delta and Omicron involved three and four haplotypes, respectively. Overlap plots showed separate patterns of emergence and decoupling for corridor (Northern and Southern Temperate) versus non-corridor (Tropics and sometimes Arctic) climatic regions (Figure 6). The 12 markers of the VOC Alpha core behaved monolithically in Arctic, Northern Temperate and Southern Temperate regions, showing a single pattern of accumulation that peaked in May 2021. In contrast, the rise of the core in the Tropics revealed a multiplicity of accumulation patterns, with at least four distinct curve types. As described above, prevalence levels of haplotype 1 markers in the Northern Hemisphere were considerably higher than those in the Southern Hemisphere. The 22 markers of the VOC Delta core also accumulated into a single peak, although the peak was broader (spanning August-November 2021) and often reached close to 100% prevalence levels in the Northern Hemisphere. Again, the rise of the core in the Tropics showed a multiplicity of accumulation patterns, with at least seven distinct curves leading to a single peak. This noisy emergence contrasts with a rather cohesive emergence of the constellation in the other climatic regions. Finally, the 32 markers of the VOC Omicron core again showed a clear distinction between the corridor and non-corridor regions. The Northern and Southern Temperate zones showed two clear steep accumulation curves that were quite variable and offset by a 2-month period. As expected, the rise of VOC Alpha occurred earlier in the Southern Temperate zone given its presumed South African origin. In contrast, the Tropic and Arctic zones showed three to four main accumulation routes, each of which had much closer origins and were less steep. A comparison of the number of non-overlapping monthly prevalence counts (symbols in Figure 5) during the November 2021 to July 2022 period revealed that the Northern and Southern Temperate regions had 48 and 57 counts, respectively, while those of the Tropics and Arctic regions had 77 and 61 counts, respectively. The difference shows higher decoupling (i.e. decreased coalescence) occurring in non-corridor regions of the world.\n\nSeparate plots describe overlaps of mutation accumulation curves for the four climate zones. Open symbols describe regions of the timeline unrelated to the VOC of reference.\n\nMutations gradually alter the proteins of the virus by introducing errors during RNA replication or repair, presumably stochastically in the viral population. Once haplotypes appear, mutations are coordinated to maintain stable protein structures through compensation, modulate protein translation and localization, or benefit their overall functionality. While haplotypes of the VOCs Alpha and Delta constellations affected (proportionally) a relatively diverse set of proteins, those of VOC Omicron were enriched in mutations impacting the S-protein. To explore this shift in more detail we constructed âprotein interaction networksâ illustrating how interactions within or between proteins in haplotypes altered protein structure and function (Figure 7). In these networks, nodes depicted proteins and links depicted interactions between them, that is, joint protein presence in a haplotype of a VOC constellation through active coordination. The node size is made proportional to the number of haplotypes harboring mutant markers affecting only one protein (e.g. haplotypes 2 and 3). The width of links is proportional to the number of haplotypes sharing the same pair of proteins. Thus, larger nodes and thicker lines of the network highlighted a more important role of proteins and their associated protein interactions at inter- and intramolecular levels.\n\nNodes are proteins and lines in the graph are protein interactions manifesting as joint protein presence in a haplotype. Node size is proportional to the number of haplotypes harboring markers that affect only one protein. Line width is proportional to the number of haplotypes sharing the same pair of proteins. Larger nodes and thicker lines highlight the significance of protein roles.\n\nHaplotype interactions of VOC Alpha (Figure 7, green lines) involved the structural S-protein and N-protein molecules, few nonstructural proteins (NSP3, NSP6, NSP12), and the ORF8 accessory protein. VOC Delta expanded the interaction repertoire by constructing two subnetworks connected by the N-protein, one involving the S-protein, M-protein, NSP12, and NSP13 and the accessory proteins ORF3a and ORF7a (mediated by haplotypes 6 and 9â) and the other one involving NSP3, NSP4, NSP6, NSP14 and ORF7b (mediated by haplotypes 7 and 10â). VOC Omicron integrated these two subnetworks with additional structural and nonstructural proteins. Note the increasing importance of the M-protein, NSP3 and NSP6 proteins, and the intramolecular interactions of the S-protein and N-protein. It is also noteworthy how interactions between structural proteins were enhanced following each VOC replacement.\n\nWe explored the location of mutations in the S-protein for two reasons: the centrality of the S-protein in viral infection and the massive enrichment of mutations in VOC Omicron. We asked if haplotype markers were spread or clustered along the S-protein sequence. Remarkably, we found two distinct behaviors (Figure 8). Markers of haplotypes 1, 12, and 14 were spread through large swaths of the sequence, especially those of haplotype 14. These haplotypes share the unique property of impacting fusogenic regions of the S2 subunit. In contrast, markers of the rest of the haplotypes were significantly clustered. For example, markers of haplotypes 4, 13, 16, and 19 localized to the RBD, while haplotypes 4 and 13 targeting exclusively the receptor-binding motif (RBM) of the domain. Similarly, markers of haplotypes 3, 7 and 8 localized to the NTD. The most recent haplotypes of VOC Omicron, haplotypes 16 and 17, were particularly interesting because they targeted both the NTD and RBD domains. We hypothesize that haplotypes with markers that are spread involve allosteric interactions that regulate the functional activities of the S-protein, including receptor-binding, membrane fusion, and interactions with other proteins. Haplotypes with clustered mutations appear to have more direct roles, impacting the binding activities of the NTD and RBD regions of the molecule. Free-standing mutations (Figure 8, labeled in grey) were particularly spread throughout the S1 subunit and often tightly associated with haplotypes linked to these regions, suggesting a tendency towards coalescence. For example, the RBM-linked S478K and Q493R markers were closely clustered with markers of haplotypes 4 and 13 while at the same time differing in patterns of accumulation (Figure 8). Mapping haplotype history also uncovered interesting trends. Except for haplotype 4, all VOC Alpha and Delta haplotypes failed to target the RBD region. Instead, the focus was flexible regions between the two subunits of the S-protein or the seasonality-linked NTD region. The arrival of VOC Omicron shifted the mutational spectrum from those regions first to RBD and then to the C-terminal region of NTD.\n\nThe diagram maps mutations onto the amino acid sequence of the S-protein molecule, from the N- to the C-terminus, with markers specific to VOCs Alpha and Delta indicated at the top and those specific to VOC Omicron at the bottom. Mutations in VOC Omicron cluster in groups according to haplotype and are enriched in immune evasion functions associated with the RBD region. Mutations in haplotypes 1, 12, and 14 spread through the molecule and likely make up networks of allosteric interactions. Clusters 1, 2, and 3 represent mutation targets at codon sites known to be either negatively selected or evolving under no detectable selection in non-Omicron sequences (Martin et al., 2022). Markers highlighted in grey represent free-standing mutations. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor-binding motif; CS, cleavage site; FP, fusion peptide; IFP, internal fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain.\n\nWe used the local ColabFold implementation of the AlphaFold2 ab initio artificial intelligence pipeline to model the atomic structure of the M-protein. Our goal was to illustrate how modeling with deep learning tools directly from amino acid sequences can help dissect the impact of mutations on the structure of viral proteins. We focused here on the M-protein because of its increasing relevance (Figure 6); an exhaustive ab initio modeling exploration of the entire SARS-CoV-2 proteome will be reported elsewhere. Since only VOCs Delta and Omicron exhibit haplotypes altering the M-protein (Figure 6), we first compared the modeled structures of the Wuhan reference strain against those of VOC Delta and Omicron strains (Figure 9A) and then dissected the individual effects of haplotypes on molecular structure (Figure 9B). Qualitative assessments were complemented with quantitative measurements of structural differences, including the calculation of TM-scores (Zhang & Skolnick, 2004) for residues in structural deviant regions. VOC Delta differs from the reference strain by one mutation, I82T of haplotype 7. Conversely, VOC Omicron differs from the reference strain by four mutations: D3G of haplotype 14, Q19E and A63T of haplotype 15, and D3N of haplotype 22â. D3G and D3N are located in an intrinsically disordered region of the molecule (Zhang et al., 2022b). All mutations were located in the N-terminal region of the molecule, but most structural effects were felt downstream in the C-terminal β-sheet domain region.\n\nThe structures of reference and mutated variants of the M-protein were modeled directly from their sequences using AlphaFold2. Their structures were then aligned, and regions exhibiting structural differences (indexed in the structural models) were further examined qualitatively by determining deviant groups and quantitatively using template modeling (TM) scores. (A) Structural alignment of M-protein molecules of the reference Wuhan strain and those typical of VOC Delta and VOC Omicron. The locations of mutations and regions with structural differences are indicated. The table describes deviant groups and TM-scores for the different regions. (B) Structural alignment of modeled molecules evaluating structural effects of mutations of the VOC Omicron constellation and related haplotypes. The left inset at the bottom shows a schematic view of M-protein domain organization mapped onto the sequence. The three transmembrane helices (TM1, TM2 and TM3) make up a bundle and multiple strands make up the C-terminal β-sheet domain. The right inset colors structural deviant regions directly onto the aligned structures.\n\nComparing the VOC Delta and Omicron molecules to those of the reference strain revealed notable structural changes (Figure 9A). Significant and equal structural divergences occurred in the linker spanning the first and second transmembrane helices of the N-terminal triple-helix that delimits the âviral envelope domainâ bundle (region 1; residues 38-43) in both VOC molecules. Similarly, more limited structural divergences affected the β-strands anchoring the terminal loop of the C-terminal âintravirion domainâ (region 2; residues 186-191). In sharp contrast, the most distal C-terminal region of the âintravirion domainâ (region 3; residues 200-222) was the most affected, especially in the VOC Omicron variant, with shortening of the last helix by one residue and reformation of the coil into a helix at the C-terminus (residues 218-220). The TM-scores for regions 1 and 3 were significantly lower than the average of the whole structure (0.936), indicating that those regions were significantly impacted by structural change. TM-scores range from 0 to 1, with 1 indicating perfect structural match and values below 0.2 indicating structural matches should be considered random (Zhang & Skolnick, 2004).\n\nThe individual effects of VOC Omicron haplotypes on the M-protein structure were even more revealing (Figure 9B). We identified nine regions with significant structural differences. Only two of these regions affected the N-terminal âintravirion domainâ and only four (regions 1, 6, 8, and 9) had TM-scores lower than the average TM-score of the entire structure (0.891). Remarkably, we observed that the VOC Omicron mutant constellation balanced the effect of the individual haplotypes. In region 1, VOC Omicron counteracted the effect of haplotype 14, which caused a conformational shift that twists the first helix of the âviral envelope domainâ at amino acid residue 21 and reduced the TM-value to 0.682. In region 6, VOC Omicron and haplotypes 15 and 22â balanced haplotype 14 and its ability to lengthen a β-strand anchoring the terminal loop of the âintravirion domainâ (matching deviant region 2 in Figure 9A). This effect reduced the TM-score to 0.873. In sharp contrast, haplotypes 14 and 22â were unable to counteract the shortening of the last helix by 1-2 residues and the reformation of the coil into a helix in regions 8 and 9 (matching deviant region 3 in Figure 9A) caused by VOC Omicron and haplotype 15. These effects were more impactful, reducing TM-scores to 0.726 and 0.185, respectively. Remarkably, the highly-conserved hinge region (residues 106-116 of region 3 in Figure 8B) was hardly affected by the mutations (especially haplotype 14), leading to a drop in the TM-score to only 0.985. This hinge controls conformational changes of M-molecules affecting virus formation during infection (Arndt et al., 2010). Overall, the mutant make-up of VOC Omicron and haplotype 15 appear major contributors to the distortion of the C-terminal âintravirion domainâ while maintaining the structure of the âviral envelope domainâ and the hinge region that connects the two domains of the molecule. Thus, mutant VOC constellations balance the more extreme effects of individual haplotypes on protein structure. A similar landscape of structural frustration, which permeates the rise of haplotypes and their make-up, exists in other viral proteins, which will be reported elsewhere.\n\nWe confirmed the validity of our AlphaFold2 predictions with experimental M-protein models recently obtained by cryo-EM (Zhang et al., 2022b). The study used two monoclonal antibodies as fiducial markers (M/Fab-E and M/Fab-B) to capture two different conformations of the M-protein in the complexes, a long form and a short form. Aligning our models to the long and short structures resulted in TM-scores of 0.693 and 0.729, GDT_TS scores of 0.606 and 0.622, and superposition RMSDs of 2.82 Ã
and 2.59 Ã
for 161 and 165 aligned Cα residues (< 5 Ã
distances), respectively (Figure 10). These scores support models with accurate topologies and very good structures for membrane proteins. Note that most structural deviations occurred in the first 30 aligned residues of the M-protein, that is, in the most N-terminal transmembrane helix that holds mutations in all VOC Omicron haplotypes. Also, note that the short form showed better alignment scores. This is particularly relevant for the functional role of the modeled structures. Tomographic and other approaches have associated the long form of the M-protein with rigidity, narrow curvature of the viral envelope as formed in the endoplasmic reticulum, and increased recruitment of S-protein and N-protein during virion assembly (Neuman et al., 2011). In contrast, the short form was associated with flexibility and decreased recruitment capabilities. This suggests that our structural predictions better match M-forms that decrease the budding and efficiency of virion assembly.\n\nThe superimposed regions with residues separated by distances less than 5 Ã
colored in red.\n\n\nDiscussion\n\nThere appears to be no âtransparentâ path of transmission from viral ancestors to VOCs nor an explanation for the massive mutant constellation that characterizes the current VOC Omicron wave of the pandemic (Kupferschmidt, 2021; Mallapaty, 2022). Indeed, the appearance of VOC Omicron in Botswana and South Africa and its fulminant and massive spread throughout the world has been both puzzling and unanticipated (Viana et al., 2022). Here we offer an explanation grounded in an analysis of about 12 million genome sequences of the virus.\n\nFirst, we show that many VOC mutations were already present in various combinations during the first wave of the pandemic and were recruited piecemeal to form increasingly large constellations. For example, more than half of S-protein markers of VOC Omicron were already present on November 2020, seeding the rise of 93% of S-protein-containing haplotypes (Table 2). These results align with a previous analysis that revealed a significant number of S-protein markers were present in VOC constellations, sometimes as novel 10-mutant combinations (Caetano-Anollés et al., 2022). Thus, haplotype primordia were already emerging very early in the evolving viral population.\n\nSecond, we show that the rise of VOC markers did not occur monolithically or in a coordinated manner worldwide. Instead, idiosyncratic patterns of marker accumulation were evident in latitude-dependent regions of the world. These distinct accumulation behaviors allowed construction of a chronology of haplotypes, all of which ultimately coalesced into VOC constellations (Figure 2). Thus, the rise of constellations resembled a process of viral emergence in which haplotypes and free-standing markers were integrated piecemeal (most likely by recombination) to facilitate infection capabilities, viral transmission, and epidemic spread. Note that virus emergence is an eco-evolutionary process that rapidly samples the genomic sequence space of the viral quasispecies while locating high-fitness combinations of mutations (Dennehy, 2017). Our previous mutation accumulation study of the viral populations of Australia also supported the emergence scenario, especially because of the very early, uncoordinated, and massive displacement of mutations of VOC Delta by those of VOC Omicron occurring in a country that had not yet reopened international and regional borders and had one of the toughest restriction policies on the planet (lockdowns, extensive contact tracing, mandatory vaccination, and strong quarantine restrictions) (Tomaszewski et al., 2022). The emergence scenario also explained the puzzling fact that VOC Omicron was first detected in samples collected November 11-16, 2021 in the African continent. However, its prevalence in Australia increased massively some weeks after being first reported in the continent on November 28-29, 2021. Physically, there could be no trans-continental path of transmission that would be so massive and efficient.\n\nThird, we dissected the VOC Omicron constellation into 16 haplotypes harboring 78 mutant markers affecting almost half of the SARS-CoV-2 proteome (14 proteins). Haplotypes were identified through coordinated patterns of marker accumulation in plots describing monthly increases and decreases of mutation prevalence along four latitude corridors of the world (Figure 2). A worldwide haplotype network illustrated how haplotypes coalesced into VOC constellations (Figure 5). Our haplotype analysis matched that of Australia (Tomaszewski et al., 2022), revealing similar haplotype recruitments, dynamic behaviors, and putative molecular interactions operating at the proteome level. It also showed increased constellation coalescence, an expected outcome given that our worldwide latitude dissection was less coarse-grained than that of a single continent in the Southern Hemisphere. Remarkably, many haplotypes were decoupled by seasonality when marker accumulation in latitude corridors failed to overlap in prevalence plots (Figure 6). This highlights the highly dynamic nature of the evolving viral population worldwide and the existence of an important seasonal effect that is genetically imprinted in the viral quasispecies.\n\nOur results are not only in line with findings for Australia (Tomaszewski et al., 2022) but also those for the United States (Tasakis et al., 2021; 62,211 genomes; January 2020âApril 2021) and England (Vöhringer et al., 2021; 281,178 genomes; September 2020âJune 2021). Results are also compatible with a worldwide longitudinal study of mutations targeting the S-protein and the NSP12 polymerase (Showers et al., 2022; 437,006 genomes; January 2020âJanuary 2021). The studies suggested that the viral genome was evolving dynamically to become more structured. In all cases, mutations accumulated in bursts, often followed by sweeps, while haplotypes and VOC constellations were generated in waves. Major global shifts in the selective landscape and possible convergence between lineages likely drove the rise of constellations (Martin et al., 2022), with viruses changing in response to hosts and mitigations (e.g. vaccination). Mutational changes were particularly exacerbated when genetic drift and âsuper spreaderâ events in a sea of mutational bursts drove infrequent mutations to prominence (Choi et al., 2020). Other explanations of VOC origin (Kupferschmidt, 2021; Mallapaty, 2022) appear less compatible with the global latitude-dependent accumulation patterns we here uncovered, including founder effects or bottlenecks occurring in single chronically-infected (Ghafari et al., 2022; Magiorkinis, 2023) or HIV-infected (Tarcsai et al., 2022) patients, origins in non-human animal hosts (e.g. mouse; Wei et al., 2021), or hidden spread in multiple hosts that would massively distribute the newly emergent variants (Ghafari et al., 2022; Magiorkinis, 2023). All of these proposals stress local rather than global mechanistic explanations. While a combination of local and global mechanisms is likely, patterns of VOC emergence may result from constraints operating worldwide at different time scales on the evolving viral quasispecies, including those imparted on proteins by mappings between the spaces of possible sequences and structures (Dennehy, 2017), of which we have limited understanding (Ferrada & Wagner, 2010). We note that the âcryptic spreadâ of SARS-CoV-2 lineages has been detected in wastewater, suggesting an evolutionary convergence (Gregory et al., 2022). This spread appears to be occurring undetected by genomic surveillance within a highly diverse genetic background.\n\nSeasonal variation impacts the persistence of living beings (Dayton, 2019). It influences the abundance and distribution of organisms and viruses through space and time. The seasonal behavior of SARS-CoV-2 has become a topic of great interest (Kissler et al., 2020), especially because seasonality could assist in formulating actionable pandemic responses. Coronaviruses are considered âwinter virusesâ and are expected to exhibit seasonal behavior (Nickbakhsh et al., 2020). In fact, significant statistical associations exist between seasonal variations and the survival and transmissibility of the virus; higher latitude, colder temperatures, and lower humidity have all been linked to higher COVID-19 incidence in local and global epidemiological analyses (e.g., Demongeot et al., 2020; Sajadi et al., 2020; Burra et al., 2021; Liu et al., 2021). Effective disease transmission appeared restricted to a 30° to 60° latitude corridor in both the Northern and Southern Hemispheres with data suggesting disease outbreaks follow those of influenza, moving across the planet along a sinuous curve parallel to the solar solstice (Hope-Simpson, 1981; Deyle et al., 2016). A molecular link between environment and physiology that would explain seasonal cycles has remained elusive despite over 150 years of research on epidemic calendars (Grenfell & BjÞrnstad, 2005; Martinez, 2018). A recent worldwide mutation prevalence study, however, revealed that mutation bursts affecting the galectin-like structure of the NTD region of the SARS-CoV-2 S-protein followed hemisphere-related patterns (Caetano-Anollés et al., 2022). Furthermore, the accumulation of haplotypes involving the S-protein and other proteins was also latitude-dependent (Tomaszewski et al., 2022). We here extend these initial analyses by uncovering significant latitude-dependent differences in VOC haplotype accumulation (Figure 2) and decoupling of core haplotypes in non-corridor latitude regions (Figure 6). These findings further support seasonal cycle-mediated molecular interactions confirming viral genetics mediates seasonal behavior.\n\nThe role of the galectin-like fold of the NTD region of the S-protein is particularly significant. Both the NTD and RBD regions recognize and bind to sugars and other host cell receptors, enabling viral attachment and infection (Pourrajab, 2021). They also present N- and O-linked glycosylation sites that act as a âglycan shieldâ to camouflage the virus from host defenses. Galectins are evolutionarily conserved glycan-binding effector proteins that regulate various processes, including cellular and extracellular interactions, pathogen recognition, and inflammation (Dings et al., 2018). These roles involve binding to the carbohydrate moieties of glycoconjugates present on cell surfaces, reducing endocytosis by forming lattices, and regulating immunity signals (Pourrajab, 2021). Galectins can either facilitate or avoid infections of a wide range of pathogens (Ayona et al., 2020). The camouflaged galectin-like structures of the S-protein likely help viral attachment and immune evasion, possibly by impersonating host galectins (Wang et al., 2020). For example, the NTD domain (but not other S-protein regions) was shown to activate human monocytes to produce cytokine cascades responsible for the acute respiratory distress syndrome (ARDS) of COVID-19 in vitro (Schroeder & Bieneman, 2022). This activation mimics the effect of host galectin-3, which facilitates activation of immune cells. It is noteworthy that administration of SARS-CoV-2 galectin-like inhibitors reportedly decreased viral loads in patients (Sethi et al., 2020). These studies stress the central role that galectins play in modulating COVID-19 infection.\n\nGalectin moieties act as environmental sensors when their activities are modulated by temperature. Recent evidence of such a role comes from an unexpected source, the coral reefs of the Pacific and Indian Oceans and the devastation caused by coral bleaching. Rising ocean water temperatures and other factors of global change disrupt the healthy symbiosis between scleractinian corals and dinoflagellates and the clearance of pathogens (e.g., Kleypas et al., 2015). A number of lectins, including galectin proteins with antimicrobial immunity functions, have been implicated in thermal and disease stress responses of coral communities (e.g. Ricci et al., 2019). Remarkably, galectins of scleractinian âcauliflowerâ corals acted as environmental sensors, recognizing and clearing coral pathogens when temperatures were optimal (25oC and 30oC) while allowing pathogen survival at lower temperatures (Wu et al., 2019). A similar temperature dependence was observed when studying the flexibility of the SARS-CoV-2 S-protein with all-atom molecular dynamics simulations (Rath & Kumar, 2020). The NTD was much more mobile than the RBD, which exhibited only a flexible RBM. Increasing temperatures made the NTD top layer residues less solvent exposed while closing the flexible RBM of the RBD in the trimer. All of these residues involved mutations highlighted in Figure 8. These conformational changes sealed the visibility of the trimeric pore, burying the receptor binding residues necessary for ACE2-binding, and inactivating (perhaps reversibly) the S-protein with temperatures above 40 °C. These results suggest that NTD binding properties depend on flexibility optimized by environmental change.\n\nCaetano-Anollés et al. (2022) studied the appearance of mutation bursts affecting the S-protein and concluded that the COVID-19 pandemic followed three successive phases, the first driven by an interplay of protein flexibility and rigidity, the second by environmental sensing, and the last phase by immune escape (Figure 1A). The timeline of haplotypes (Figure 2) and their clustering along the S-protein sequence (Figure 9) support such an interpretation. The first haplotypes impacted the flexibility of the S-protein. The D614G mutation of haplotype 5 disturbed hydrogen bonding interactions between the S1 and S2 subunits of different protomers of the S-protein as well as contacts with the FP region that are necessary for membrane fusion (Korber et al., 2020; Yurkovetskiy et al., 2020; Xu et al., 2021). Following the rise of haplotype 2, which affected the intrinsically disordered linker region of the N-protein (Tomaszewski et al., 2020), haplotype 1 introduced four S-protein markers (A570D, T716I, S982A and D1118H) in regions of increased protein disorder that mostly affected the C-terminal S2 subunit (Caetano-Anollés et al., 2022). Haplotype 3 introduced deletions (H69del, V70del, and Y144del) located in the NTD region holding the galectin-like structure associated with environmental sensing and seasonal behavior (Caetano-Anollés et al., 2022). Finally, haplotype 4 and a free-standing mutation introduced two crucial markers (N501Y and P681H) impacting the immunogenic RBD region responsible for ligand binding (e.g., ACE2). Note that N501Y involved one of six contact residues of RBD known to increase both ACE2 receptor affinity and virulence (Starr et al., 2020), while P681H altered one of four residues comprising the insertion that creates the S1/S2 furin cleavage site between the S1 and S2 subunits (see Harvey et al., 2021). VOC Delta haplotypes appearing at the beginning of the immune escape phase focused for the most part on mutations in other proteins but continued to involve the environmental sensing NTD region (T19R of haplotype 7 and deletions E156G, F157del, and R158del of haplotype 8) and the RBD site (P681R marker of haplotype 7). Similarly, free-standing markers also impacted the NTD (T96I, G142D, A222V) and RBD (L452R, T478K) regions. VOC Omicron recruited most of these markers via haplotypes 3, 4, and 12 but then massively acquired mutations in the RBD, NTD and S2 subunit regions via several additional haplotypes, with the novelty that two of these haplotypes affected both the sensing and immunogenic regions (haplotypes 16 and 17) and one affected all three regions (haplotype 14) (Figure 8). Thus, tight networks of intramolecular interactions appeared to unify change in all functional regions of the S-protein as the pandemic advanced. Note that the entire constellation of VOC Omicron is under gene-wide positive selection (Viana et al., 2022) and that many mutations arose from molecular interactions that were collectively adaptive (Martin et al., 2022). A group of 13 was clustered into three regions of the S-protein (horizontal lines in Figure 8) mapping to haplotypes, 12, 13, 16, and 19.\n\nNetworks of protein interactions describe haplotype-mediated protein communications that impact the structure and function of proteins (Figure 7). Typically, these molecular interactions involve direct communications within (e.g., allosteric control) or between molecules (e.g., protein-protein interactions), or indirect communications through shared or linked functions. We traced how protein networks changed with every VOC replacement to study evolutionary constraints and pathways of evolutionary optimization. As mutant constellations evolved, protein interaction networks became organized around the S-protein, N-protein, and M-protein molecules via intramolecular and intermolecular relationships (Figure 7). This is an expected outcome. Intraviral interactions between these three structural proteins are essential for hijacking the hostâs cellular machinery (Siu et al., 2008; Fehr & Perlman, 2015). The multifunctional spike glycoprotein plays roles in target recognition (e.g. viral attachment to cell receptors, cellular tropism), cellular entry (viral fusion), and endosomal escape (e.g., capsid assembly), not to mention roles in transmissibility and virulence (Huang et al., 2020; Magazine et al., 2022). Its highly immunogenic properties have made the S-protein a target for drug and vaccine development (Harvey et al., 2021). The N-protein packages the RNA genomes but plays critical roles in replication, virion assembly, and regulation of the viral life cycle (Bai et al., 2021). Proteolysis of the intrinsically disordered linker that separates the two domains of the N-protein generates at least five proteoforms that bind structured RNA and provide regulatory and immunogenic functions (Lutomski et al., 2021). The transmembrane M-protein is crucial for virus assembly and membrane budding (Fehr & Perlman, 2015). It consists of an N-terminal âviral envelopeâ ectodomain made of three transmembrane helices and a C-terminal globular âintravirionâ endodomain. The endodomain interacts with the N-protein, S-protein and RNA molecules for oligomerization, RNA encapsulation, and mature virus particle formation but also with the E-protein with the help of the two most central transmembrane helices (Hsieh et al., 2008; Zhang et al., 2022b). The M-protein localizes in the endoplasmic reticulumâGolgi intermediate compartment and recruits other viral structural proteins (Fehr & Perlman, 2015). Molecular dynamic and docking simulations of SARS-CoV-2 structural proteins recently revealed that the M-protein acts as a receptor, while the S-protein, N-protein, and E-protein act as protein ligands (Kumar et al., 2021). This is in line with cryo-EM, tomography and statistical evidence supporting the central role played by the M-protein in virion assembly (Neuman et al., 2011). Remarkably, all of these interactions materialize in the evolving haplotype-mediated interaction networks.\n\nSimilarly, domains and linkers of the N-protein interact with a number of proteins, including the M-protein (Lu et al., 2021) and the multidomain NSP3 papain-like protease (Hurst et al., 2013). NSP3 processes viral polyproteins, forms the viral replicase-transcriptase complex with other NSPs and RNA, and antagonizes the host innate immune response (Lei et al., 2018). The N-protein and NSP3 connection was established early via multiple markers of haplotype 1 in VOC Alpha, haplotype 6 in VOC Delta, and haplotype 16 in VOC Omicron (Figure 7). The functionalities of the S-protein, nucleocapsid and NSP3 proteins coalesce in the VOC Omicron network, highlighting their well-known centrality in viral transmissibility, disease severity, and immune escape. Their role is further enhanced by interactions with the autophagy-associated NSP6 protein. NSP6 induces formation of multimeric sensor proteins (inflammasomes) and autophagosomes, mediating caspase-1 activation and secretion of pro-inflammatory cytokines known to induce inflammatory cell death (Cottam et al., 2011; Sun et al., 2022). Aberrant activation of inflammasomes can cause cascades leading to the severe respiratory syndromes of SARS-CoV-2 (Rodrigues et al., 2021). NSP6 network connections were established early via haplotype 4 in VOC Alpha and then VOC Omicron, haplotype 6 in VOC Delta, and haplotype 14 in VOC Omicron (Figure 7). This prompts evaluation of how the new constellations are softening aberrant immunity activations.\n\nThe effect of mutations on the protein sequence must be linked to effects at atomic structure level to dissect the functional significance of individual haplotypes and constellations. Three main strategies model protein structure: homology modeling, fold recognition, and ab initio methodologies. Homology modelling and fold recognition rely on the existing sequence and folded structure data and are rather comparative in nature. These methods can be limited in their ability to accurately predict the true 3-dimensional structure of novel proteins, especially in molecular systems subjected to fast mutation rates. Ab initio methods however do not use pre-existing knowledge. Instead, they build models directly from amino acid sequences and the stoichiometric constraints of those sequences. Such an approach is especially useful for modeling proteins with low homology. We modeled the 3-dimensional atomic structures of mutant SARS-CoV-2 proteins defining haplotypes and constellations with AlphaFold2 (Jumper et al., 2021). AlphaFold2 is the star of the last two biannual structure prediction experiments (CASP 14 and 15). Its deep learning algorithm makes fast atomic structural predictions with levels of accuracy that are within the margin of error of experimental structure determination methods. Crucially, this reduces reliance on traditional crystallographic methods that are time-consuming. Because of the central role that the M-protein plays in delimiting protein interactions (Figure 6), we here report the effects of mutations on its structure (Figure 9). While mutations were located in the N-terminal region of the molecule, most structural effects were felt downstream in the C-terminal âintravirionâ endodomain responsible for interactions with other structural proteins and their recruitment for virion assembly (Fehr & Perlman, 2015). The mutant make-up of VOC Omicron and haplotype 15 appear to be major contributors to endodomain distortions, but both maintained the structure of the transmembrane âviral envelopeâ domain and the hinge region that connects the two domains of the molecule. In sharp contrast, haplotype 14 twisted the first transmembrane helix. The hinge, which adopts a helix-turn structure inserted into a cavity formed by the triple-helix transmembrane bundle, is a key element for the conformational change and M-protein function, including viral assembly, mediating transition between two conformational states that are in equilibrium (Zhang et al., 2022b). In fact, mutations and deletions in the hinge region are known to inhibit virus formation (Arndt et al., 2010), explaining the structural conservation of the hinge. Thus, modeling the proteins affected by haplotypes and VOCs confirms the functional centrality of this region. One remarkable finding of these ab initio modeling exercises and others we will report elsewhere is that VOC constellations counteracted the more extreme effects of individual haplotypes on protein structure. This strongly suggests that a cooperative activity exists in protein communications that was made explicit in our protein interaction networks. This information will be especially valuable for therapeutic interventions and predictive intelligence applications. They could facilitate understanding of protein reformation, de novo protein and construct design, and formation of molecular complexes.\n\n\nConclusions\n\nOur study uncovers a rationale behind the noisy and dynamic emergence of VOCs and their increasingly complex makeup. While the SARS-CoV-2 genome is evolving in bursts, recombination and recruitment processes gradually generate a number of haplotypes, some of which coalesce into apparently monolithic constellations. Remarkably, seasonal effects decouple these constructs showing they are highly dynamic. Thus, viral evolution appears attuned to the seasonal periodicities of the planet that arise from Earthâs tilted axis of rotation thanks to molecular sensors that probe environmental change and genetically link the structure and function of proteins.",
"appendix": "Data availability\n\nGISAID EpiCoV⢠(https://gisaid.org): COVID-19 genomic dataset and metadata associated with 11,921,113 sequences available under GISAID identifier EPI_SET_230208zs\n\nhttps://doi.org/10.55876/gis8.230208zs. Access to the data requires registration and agreement to the conditions for use at: https://www.gisaid.org/registration/register/.\n\nModelArchive: AlphaFold2 structural predictions under accession ma-gca-mprot: https://dx.doi.org/10.5452/ma-gca-mprot\n\nThe ModelArchive data are available under the terms of the Creative Commons Attribution-ShareAlike 4.0 International license (CC BY-SA 4.0).\n\nbioRxiv: Supplementary Table 2 of Showers et al. (posted on March 05, 2021): https://doi.org/10.1101/2021.03.05.433666\n\nThe Showers et al. data are available under the terms of the Creative Commons Attribution-NoDerivatives 4.0 International license (CC BY-ND 4.0).\n\nZenodo: Seasonal effects decouple SARS-CoV-2 haplotypes worldwide. https://doi.org/10.5281/zenodo.7636393 (Tomaszewski et al., 2023)\n\nThis project contains:\n\n- The list of 183,276 mutations\n\n- The complete list of accession IDs\n\n- The GISAID acknowledgement file.\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgments\n\nWe gratefully acknowledge all data contributors, i.e., the authors and their originating laboratories responsible for obtaining the specimens, and their submitting laboratories for generating the genetic sequence and metadata and sharing via the GISAID Initiative, on which this research is based.\n\n\nReferences\n\nArndt AL, Larson BJ, Hogue BG: A conserved domain in the coronavirus membrane protein tail is important for virus assembly. 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}
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[
{
"id": "168134",
"date": "04 Apr 2023",
"name": "Francisco Solano Muñoz",
"expertise": [
"Reviewer Expertise Molecular Biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an impressive, long but excellent review about worldwide SARS-CoV-2 evolution. The effect of a number of factors are widely discussed, with nice figures and reasonable classifications. I have spent a long time in reading the article, as I was interested in that and I have learned with the reading. The introduction is nice and clear, containing simple and helpful definitions to follow the manuscript. This is formative and didactic. I encourage the authors to define the term 'clade' in relation to variant and constellations. Another minor point would be at Page 4, concerning the sentence âThe first VOCs of SARS-CoV-2 appeared in October 2020, few months after the first wave of the pandemicâ. This VOC should be identified and referenced.\nI have no objections but only some questions or curiosities. I recognize that it is a little bit contradictory to report that the review is too long and at the same time to ask for clarifying some points. I hope the authors will be able to conjugate both points.\nThis review is in agreement with other studies of the same group, as the AustraliaÂŽs study, but similar studies from other groups are not discussed. In that way, the review is somehow considered an extension of the Australia study form and endogamic point of view. In that way, for instance, a brief comparison to some recent similar study would be welcome. I propose Cairo et al. (20221).\n\nIt is stated that only three out of five VOCs, Alpha, Delta and Omicron, became predominant worldwide at some point, completely replacing each other. Beta and gamma VOCs, probably initiated in Brazil and South Africa, have not been considered in this study. Any brief data or discussion about beta and gamma would be welcome. The main reasons why they were not so predominant would be briefly discussed. On the other hand, are they related to some of the 20 haplotypes describes in this review? Do the 20 haplotypes comprise all the worldwide pandemic evolution?\n\nPlease, define viral quasispecies.\n\nThe review dissected the Omicron constellation into 16 haplotypes harboring 78 mutant markers affecting almost half of the SARS-CoV-2 proteome (14 proteins). I wonder why 14. The criteria for choosing 14 proteins and not the whole viral proteome should be described.\n\nThe Discussion is too long indeed. Some paragraphs in the discussion are not related with the title.\n\nThe Title emphasizes the seasonal effects in a so extensive study. I recommend changing the title or reduce the results and discussion.\n\nFigures 9 and 10 are not directly related to the main objectives of the review. They could be omitted in order to reduce space. I leave this point to the editor and author`s consideration, but the review is too long for other Journals that I have reviewed for. I have no experince in F1000Research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "168135",
"date": "14 Apr 2023",
"name": "Kyung Mo Kim",
"expertise": [
"Reviewer Expertise Bioinformatics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aims to investigate the evolutionary history and mechanisms of SARS-CoV-2 viral populations. The authors provide sufficient background information, such as the clinical impact, mutability, evolutionary forces, and infection mechanisms, to help readers understand the remaining parts of the manuscript. By analyzing about 12 million SARS-CoV-2 genomes using phylogenomic and statistical techniques, the authors documented valuable findings in this paper.\nThe phylogenetic analysis based on amino acid changes between the genomes showed the emergence of mutants, haplotypes, and VOCs and their evolutionary relationships to each other. Through the prevalence analysis, the authors identified mutants that contribute to the temporal and spatial changes of VOCs, detected decoupling, and newly defined mutant constellations for VOCs. These findings must be valuable for therapeutic interventions against this virus. Additionally, it is worth noting that many VOC mutations appeared during the early phase of the pandemic and that these early-emerging mutations were recruited to form major haplotypes and VOC mutant constellations. The authors further discovered that haplotypes can be unified or diversified by a combination of recruitment, coalescence, decoupling, and loss. Finally, the ab initio modeling produced an interesting result that haplotype divergence resulting in protein structural variations can be balanced by the mutant constellations.\nOverall, this manuscript is well-written, but there are a few minor points to consider. Firstly, it would be helpful to explain the GISAID standards for \"high-coverage.\" Additionally, the authors may provide a rationale for why they only analyzed substitutions with sequence counts greater than or equal to one standard deviation over the mean of the entire sequence set. It might also be worth considering to use the standard deviation of 1.96 that corresponds to a 95% confidence interval. Secondly, the word \"accumulation plot\" may not be appropriate since a decrease (loss) of mutation prevalence is often observed. I think that âprevalence plotâ can be used throughout the text. Finally, the first few sentences in the Molecular Structure section of the Discussion that describe some major strategies for protein structure modeling could be moved to the Introduction or removed from the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-267
|
https://f1000research.com/articles/11-1456/v1
|
08 Dec 22
|
{
"type": "Research Article",
"title": "Evaluation of vitamin D supplementation intake among children; cross-sectional observational study",
"authors": [
"Niloufar Sharafi",
"Aiman Fatima",
"Syed Wasif Gillani",
"Nour Kaddour",
"Rawa Banoori",
"Riham Mohamed Elshafie",
"Hassaan Anwer Rathore",
"Niloufar Sharafi",
"Aiman Fatima",
"Nour Kaddour",
"Rawa Banoori",
"Riham Mohamed Elshafie",
"Hassaan Anwer Rathore"
],
"abstract": "Background: The purpose of this study was to review the vitamin D supplementation intake status among children in the general public, determine the vitamin D supplements practices, and the barriers that parents and children face with supplementation.\n\nMethods: A cross-sectional observational questionnaire-based survey study design was used. A convenience sampling technique was used to collect the data. An online Rao soft sample size calculator was applied to determine the sample size of 319. The response rate of participants was expected to be 63%, the margin of error was 5% and the level of confidence was 95%.\n\nResults: A total of 248 parents (89.1% mothers (n =203)) and 15.7% fathers (n=39) with a mean ± SD age of 35.4 ± 7.04 years, completed the study (77.7% response rate). Parents reported that the supplements used the most by children were vitamin D supplements (21.85%) and multivitamins (21.8%) followed by calcium supplements (5.6%). However, 27.8% of children in this study did not take any supplements. Of all the parents, 65% (162) of them reported sending their child outside to play while 34.67% (86) of parents had reported no outdoor activity. Approximately 184 (74.2%) parents reported the childâs diet to contain multiple natural sources of vitamin D. However, 69 (27.8%) parents reported giving none of the natural sources of vitamin D to their children through the diet. Parents with higher education about 62.9% (n=156) had a higher frequency of providing vitamin D supplements to their children. Children in high-income families (43.63%) were more likely to take vitamin D supplements than those in middle- or low-income families.\n\nConclusion: The study concluded that challenges like the educational and financial background of parents, family-income level, and health insurance status could help aid in addressing the overall burden of vitamin D deficiency among young children.",
"keywords": [
"Vitamin D",
"deficiency",
"frequency",
"supplementation",
"dietary sources",
"sunlight exposure",
"challenges"
],
"content": "Introduction\n\nVitamin D is known among the critical minerals to play an important role in maintaining normal body functions.1 It allows bone mineralization and avoids hypocalcemic tetany (such as involuntary muscle contraction, cramps, spasms, etc).2 It is also known for aiding osteoblasts and osteoclasts in developing and remodeling the bone preventing it from being brittle.3 Other functions of vitamin D in the body include inflammation reduction and regulation of cell growth, neuromuscular and immune function, and glucose metabolism.4 Vitamin D also affects the expression of several genes that code for proteins that govern cell proliferation, differentiation, and apoptosis. Vitamin D receptors can be found in many tissues, and some of them transform 25(OH) D to 1,25 (OH) D.5\n\nMaintaining optimum levels of calcium and vitamin D during childhood and adolescence is critical for bone growth.6 Vitamin D is said to lower the risk of cancer, prevent viral infections, alleviate musculoskeletal pain, and calm mood disorders including depression, according to some claims. There has also been a surge in scientific interest in studying vitamin D at both the basic and clinical levels to address these and other claims.7 Children with vitamin D deficiency develop a disease known as rickets, which is characterized by a frame and fragile bone, making the legs appear bent.8 Vitamin D has been shown to reduce the risk of premature birth in pregnant women.9\n\nA child's vitamin D deficiency can start as early as birth, which can damage not just their bone metabolism but also their immunological system, making them more susceptible to illnesses early in life.10 For the treatment of vitamin D deficiency rickets, the American Academy of Pediatrics (AAP) recommends an initial two- to three-month regimen of âhigh-doseâ vitamin D therapy of 1000 units daily in neonates, 1000 to 5000 units daily in infants one to 12 months old, and 5000 units daily in patients over 12 months old.11\n\nEpidemiologic studies, at least in adults, apart from the risk of osteomalacia and osteoporosis, have associated hypovitaminosis D with an increased risk of several cancers, autoimmune diseases (type 1 diabetes, multiple sclerosis, rheumatoid arthritis, and Crohnâs disease), heart disease, hypertension, metabolic syndrome, asthma, upper respiratory tract infections, muscle weakness, and falling.12 The pleiotropic action of vitamin D was already revealed on molecular, cellular, tissue, and organ levels.13 These observations modified the current knowledge about vitamin D metabolism and methods of diagnosis of vitamin D deficiency states.14\n\nUnfortunately, vitamin D is found rare in food.15 Vitamin D is found in only a few foods. Fish liver oils and the meat of fatty fish (such as trout, salmon, tuna, and mackerel) are among the greatest sources. The amount of vitamin D in a human's tissue is influenced by its food. Vitamin D is also found in modest levels in beef liver, egg yolks, and cheese, mostly in the form of vitamin D3 and its metabolite 25(OH)D3.16 Vitamin D2 is found in varying levels in mushrooms. Some commercially available mushrooms have been exposed to UV radiation to boost their vitamin D2 levels. In addition, the FDA has approved UV-treated mushroom powder as a food additive for use as a vitamin D2 source in food items.17\n\nFurthermore, vitamin D is added to milk, many ready-to-eat bowls of cereal, and some yogurt and orange juice brands. It is found in modest concentrations in cheese and some margarine.18\n\nIn the U.A.E., the consistent predominant hot weather, inadequate exposure to sunlight, and low nutritional intake of vitamin D result in low serum concentrations of circulating 25(OH) D, a condition known as hypovitaminosis D.19 Furthermore, recent lifestyles involving using cars for transport over walking, and children indulging in electronics and staying indoors have also influenced low vitamin levels. Low dietary intake of vitamin D and calcium, and other factors, including obesity and low social status, are all associated with low serum levels of vitamin D.20â22\n\nFurther research is needed to be conducted on the production of high-potencyâfood-based vitamin D supplements, the move to mandatory fortification of cereal grain staples, and the development of natural food sources with higher vitamin D content are all potentially safe and efficient pathways for overcoming the barriers to optimal vitamin D status. Although various studies suggest a high prevalence of vitamin D deficiency among adults and children, no randomized controlled trials have been fully performed on vitamin D deficiency and supplementation among children in the UAE. When compared to the expense of providing therapies for many chronic diseases closely linked to vitamin D deficiency, taking vitamin D supplements is a much better alternative.\n\nThe purpose of this study was to review the vitamin D supplementation intake status among children in the general public, to evaluate the vitamin D supplements practices, the natural sources of vitamin D from their diet, and the barriers that parents and children face with supplementation.\n\n\nMethods\n\nA cross-sectional observational questionnaire-based survey study design was used in this study. The survey was conducted in public places in the U.A.E. The data was collected over seven months from October 2021 to April 2022.\n\nThe questionnaire was adapted from the literature and evaluated for content validation.\n\nPart one of the questionnaire consists of demographic information (e.g. age, nationality, gender, etc.)\n\nThe second part collected information about supplementation intake and natural sources of vitamin D intake (e.g, if the child has milk, yogurt, etc. in his diet).\n\nThe last part consists of the outdoor activity level of the child (e.g. hours the child spends playing outdoors).\n\nAll the parts of the data were collected based on yes or no or multiple response questions.23\n\nVitamin D deficiency, education level of parents, outdoor activity hours of children, health insurance status of parents, income level of the families.\n\n\n\n⢠The primary outcome of this study was to observe the vitamin D supplementation intake status.\n\n⢠The possible reasons for vitamin D deficiency among children.\n\nParents may have difficulties in supplementing their children's diets due to their financial level, health insurance status, and level of literacy.\n\nA convenient sampling technique was used to collect data from approximately 248 participants from public places in Ajman, U.A.E. An online Rao soft sample size calculator was applied to determine the sample size, which was 319. The response rate of parents was expected to be 63%, the margin of error was 5% and the level of confidence was 95%.24 Around 319 participants were expected and 248 responded and actively participated in the study, 77.7% was the response rate of the study participants.\n\nThis study has no missing data.\n\nParticipants who had at least one child between 4-15 years of age and who agreed to participate.\n\nChildren with minor illnesses that are common in the general population and those suspected clinically of having rickets. Children with cognitive and behavioral disorders were excluded from the study.\n\nEthics approvals have been obtained for the study. This is the ethics approval number IRB/COP/STD/74/Oct-2021 from GMU.\n\nThe questionnaire content was described before giving it to the parents and the written consent form was taken from each participant. The consent form was as follows:\n\nâYour participation in this survey is voluntary. You may choose not to participate. If you decide to participate in this survey, you may withdraw at any time. If you decide not to participate in this study, or if you withdraw from participating at any time you will not be penalized. Filling out this form means that you accept to participate in this research.â\n\nThe data analysis was done using the SPSS statistical package for social sciences software. A Chi-square analysis was done. Mean, standard deviation, and mean comparison was utilized for continuous data. Both a tabular and graphic version of the data was used to show it. A 5% degree of confidence and a 0.5 margin of error were chosen.\n\nThere is no bias in any trend in the collection, analysis, interpretation, or review of the data that can lead to conclusions that are different from the truth.\n\n\nResults\n\nThe study reported no missing data. A total of 248 parents (89.1% mothers (n=203)) and 15.7% fathers (n=39) with a mean±SD age of 35.4±7.042 years, completed the study. About 62.9% (n=156) parent were holding a university degree, while 26.2% (n=65) of the participants completed a secondary school, 9.7% (n=24) completed primary school and 1.2% (n=3) of parent were uneducated. Moreover, almost 61.7% (n=153) of mothers were unemployed, while a smaller number of mothers 27% (n=67), and 11.3% (n=28) were employed and employed with medical background. For fathers the 70.2% (n=174) were employed and 21% (n=52), 4.8% (n=12) and 4% (n=10) were self-employed, employed with a medical background and unemployed, respectively. Approximately 42.7% (n=106) participants had income level of more than 10,000, while 35.5% (n=88), 10.1 (n=25) and 4.8 (n=12) had income level of 5,000-10,000, 2,000-5,000 and less than 2,000, respectively. More than half of the participants has insurance and only a few participants 28.6% (n=71) had no insurance (Table 1).\n\n* Caregivers.\n\nThis research has participants from different countries (total=23). The majority (67%) of participants are from five countries (India, Iran, Pakistan, Syria, and Emirates). The completed data has been presented in Figure 1.\n\nA higher proportion of children received supplements whose parents were educated to the level of secondary school and above. Table 2 shows the literacy level of parents and supplementation.\n\nThe data on outdoor activity levels included the average frequency of outdoor activity per day. Of all the parents, 65% (162) of them reported sending their child outside to play while 34.67% (86) had no outdoor activity. The mean hours of outdoor activity for the children were 2.046±1.61. It was found that on average, children spent 0.15-6 hours playing outside in the sun therefore exposed to sunlight (Table 3).\n\nParents reported that supplements used the most by children were Vitamin D supplements (21.85%) and multivitamins (21.8%) followed by calcium supplements (5.6%). However, 27.8% of children in this study did not take any supplements. While other parents reported mixed intake of supplements (for example some children took vitamin D plus calcium supplements while others took vitamin D and multivitamins (Figure 2).\n\nFigure 3 summarizes and describes that out of the 248 participants, 184 (74.2%) parents reported their childâs diet to contain multiple natural sources of vitamin D (for example some children had milk plus cheese in their diet while others had yogurt plus cheese plus vitamin D fortified orange juice). However, 69 (27.8%) parents reported giving none of the natural sources of vitamin D to their children through the diet.\n\nChildren in high-income families (43.63%) were more likely to receive vitamin D supplements than those in middle- or low-income families (Table 4).\n\nParents with private health insurance 51.85% were more likely to give vitamin D supplements to their children compared to those with government health insurance 25.92% and no health care insurance 22.22% (Table 5).\n\nThe study included participants from 23 various nationalities. The top five countries with the most participants were, India, Pakistan, Syria, and the U.A.E. Out of 34 participants from India, only 11 (32.35%) reported the use of vitamin D supplementation by their children, four (11.76%) reported using calcium supplements, six (17.64%) reported using multivitamins and the other 6 of them (17.64%) reported giving multiple sources of vitamins to their children.\n\nHowever, out of the 34 participants from India, seven (20.58%) did not use any vitamin supplements at all. While out of 28 participants from Iran, only 6 participants, (21.41%) reported the use of vitamin D supplementation by their children, three (10.71%) reported using calcium supplements, ten (35.71%) reported using multivitamins and the other eight of them (28.57%) reported giving multi-sources of vitamins to their children. Among the 34 participants from Pakistan, the lowest amount of vitamin D supplements intake, only four (11.76%) participants reported giving supplements to their children and three (8.82%) reported using calcium supplements, only one (2.94%) parent reported using multivitamins and the other 12 of them (34.29%) reported giving multi-sources of vitamins to their children. Out of 27 participants from Emirates, few participants which are five (18.51%) of them reported the use of vitamin D supplementation by their children, two (7.4%) reported using calcium supplements, seven (25.94%) reported using multivitamins and the rest 11 (40.74%) reported giving multi-sources of vitamins to their children. The participants from Syria reported the highest vitamin D intake among the countries, out of 40 participants from India, only 12 (30.76%) reported the use of vitamin D supplementation by their children, one (2.56%) reported using calcium supplements, nine (23.07%) reported using multivitamins and the other 10 (25.64%) reported giving multiple sources of vitamins to their children Table 6.\n\n\nDiscussion\n\nThis cross-sectional study demonstrates that inadequacy of vitamin D remains a risk in the U.A.E due to its geographical location being at the equator leading to the hot harsh climate throughout the year limiting the childrenâs exposure to sunlight. Depending on the effectiveness of UVB photons to promote vitamin D production, the amount of sun exposure necessary for the creation of ultraviolet B-induced vitamin D in the skin impacts cutaneous synthesis.25 This study also identifies the association between socioeconomic and demographic barriers with vitamin D supplementation among children. Children in high-income families (43.63%) were more likely to take vitamin D supplements than those in middle- or low-income families.\n\nParents with private health insurance (51.85%) were more likely to provide vitamin D supplements to their children compared to those with government health insurance (25.92%) and no health insurance (22.22%).\n\nFurthermore, it was found in this study that parentsâ education and literacy level largely determines children receiving vitamin D supplementation. Parents in this study with a lower literacy rate who did not complete primary school were less likely to give vitamin D supplements to their children. However, evidence of gender differentials in vitamin D supplementation was not found in this study.\n\nIn U.A.E., since there is no law requiring the fortification of vital foods with vitamin D, there are few vitamin D-fortified products on the market.26 Among the participants of the study, 184 (74.2%) reported the childâs diet to contain multiple natural sources of vitamin D (for example some children had milk plus cheese in their diet while others had yogurt plus cheese plus vitamin D fortified orange juice) and most parents reported that their child has milk in their diet. Moreover, 69 parents (27.8%) reported giving none of the natural sources of vitamin D to their children through the diet. As a result, individual vitamin D dietary intake is strongly influenced by dietary preferences as well as the country's fortification plan. Without supplementation, however, vitamin D status is heavily on endogenous vitamin D synthesis, which is influenced by genetic determinants and lifestyle.27\n\nVitamin D aids calcium absorption in the intestine by facilitating active calcium transport across the mucosa. Vitamin D insufficiency is usually caused by a lack of calcium in the diet and leads to bone deterioration or osteoporosis.28 The results of this study are generalizable since it has been done on general population and multiple ethnicities in the U.A.E, it reflects that this study is generalizable to different parts of the world.\n\nHowever, this study had potential limitations. From a methodological point of view, the weakness of the study is that it is based on a cross-sectional design. The inherent problem of a cross-sectional design is that the outcome (vitamin D supplementation status) and the exposure (in this case, socioeconomic characteristics and a Stateâs social and economic development status) are collected simultaneously, thereby preventing conclusions regarding causality. The data was mostly collected from mothers. The present literature lacks data on the vitamin D level of children, thus a comparison between outdoor level activity of the children and the vitamin D level of children was presented in this study.\n\n\nConclusion\n\nThe findings of the study concluded that the educational and financial background of parents and health insurance status could aid in addressing the challenges parents face with providing vitamin D supplements to their children as well as nutritional assessment for early natural supplement treatment.",
"appendix": "Data availability\n\nFigshare. Parents reported Vitamin D Supplementation among Children (Responses). DOI: https://doi.org/10.6084/m9.figshare.20207165.v1. 29\n\nThis project contains the following data:\n\n- The purpose of this study was to review the vitamin D supplementation intake status among children in the general public, determine the vitamin D supplements practices, and the barriers that parents and children face with supplementation.\n\n\nReferences\n\nDeLuca HF: The metabolism and functions of vitamin D. Steroid Hormone Resistance. 1986; pp. 361â375. Publisher Full Text\n\nBikle DD: Vitamin D and bone. Curr. Osteoporos. Rep. 2012 Jun; 10(2): 151â159. PubMed Abstract | Publisher Full Text\n\nFeng X, McDonald JM: Disorders of bone remodeling. Annual Review of Pathology: Mechanisms of Disease. 2011 Feb 28; 6: 121â145. PubMed Abstract | Publisher Full Text\n\nFleet JC, DeSmet M, Johnson R, et al.: Vitamin D, and cancer: a review of molecular mechanisms. Biochem. J. 2012 Jan 1; 441(1): 61â76. Publisher Full Text\n\nBikle DD: Vitamin D: production, metabolism, and mechanisms of action. Endotext. 2021 Dec 31.\n\nCashman KD: Vitamin D in childhood and adolescence. Postgrad. Med. J. 2007 Apr 1; 83(978): 230â235. PubMed Abstract | Publisher Full Text\n\nWeydert JA: Vitamin D in childrenâs health. Children. 2014 Sep; 1(2): 208â226. PubMed Abstract | Publisher Full Text\n\nSahay M, Sahay R: Ricketsâvitamin D deficiency and dependency. Indian J. Endocrinol. Metab. 2012 Mar; 16(2): 164â176. PubMed Abstract | Publisher Full Text\n\nKassai MS, Cafeo FR, Affonso-Kaufman FA, et al.: Vitamin D plasma concentrations in pregnant women and their preterm newborns. BMC Pregnancy Childbirth. 2018 Dec; 18(1): 1â8.\n\nBattersby AJ, Kampmann B, Burl S: Vitamin D in early childhood and the effect on immunity to Mycobacterium tuberculosis. Clin. Dev. Immunol. 2012 Jan 1; 2012.\n\nLee JY, So TY, Thackray J: A review on vitamin d deficiency treatment in pediatric patients. J. Pediatr. Pharmacol. Ther. 2013 Oct; 18(4): 277â291. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGröber U, Spitz J, Reichrath J, et al.: Vitamin D: update 2013: from rickets prophylaxis to general preventive healthcare. Dermato-endocrinology. 2013 Jun 1; 5(3): 331â347. PubMed Abstract | Publisher Full Text\n\nLai YH, Fang TC: The pleiotropic effect of vitamin D. International Scholarly Research Notices. 2013; 2013.\n\nDominguez LJ, Farruggia M, Veronese N, et al.: Vitamin D sources, metabolism, and deficiency: available compounds and guidelines for its treatment. Metabolites. 2021 Apr; 11(4): 255. PubMed Abstract | Publisher Full Text\n\nAlFaris NA, AlKehayez NM, AlMushawah FI, et al.: Vitamin D deficiency and associated risk factors in women from Riyadh, Saudi Arabia. Sci. Rep. 2019 Dec 30; 9(1): 1â8.\n\nRoseland JM, Phillips KM, Patterson KY, et al.:Feldman D, Pike JW, Bouillon R, et al., editors. Vitamin D in foods: An evolution of knowledge. 41â78.\n\nFood US, Administration D: Food additives permitted for direct addition to food for human consumption; vitamin D2 mushroom powder. Fed. Regist. 2020; 85: 41916â41920.\n\nU.S. Food and Drug Administration:January 4, 2018.Reference Source\n\nMuhairi SJ, Mehairi AE, Khouri AA, et al.: Vitamin D deficiency among healthy adolescents in al ain, united Arab emirates. BMC Public Health. 2013 Dec; 13(1): 1â7. Publisher Full Text\n\nAl-Othman A, Al-Musharaf S, Al-Daghri NM, et al.: Effect of physical activity and sun exposure on vitamin D status of Saudi children and adolescents. BMC Pediatr. 2012 Dec [cited 2021 Mar 11]; 12(1): 589. PubMed Abstract | Publisher Full Text\n\nHirani V, MosdÞl A, Mishra G: Predictors of 25-hydroxyvitamin D status among adults in two British national surveys. Br. J. Nutr. 2008 Jul 17 [cited 2021 Mar 11]; 101(5): 760â764. PubMed Abstract Reference Source\n\nGiovannucci E, Liu Y, Rimm EB, et al.: Prospective Study of Predictors of Vitamin D Status and Cancer Incidence and Mortality in Men. JNCI J. Natl. Cancer Inst. 2006 Apr 5 [cited 2021 Mar 11]; 98(7): 451â459. PubMed Abstract | Publisher Full Text Reference Source\n\nNucci AM, Russell CS, Luo R, et al.: The effectiveness of a short food frequency questionnaire in determining vitamin D intake in children. Dermato-endocrinology. 2013 Jan 1; 5(1): 205â210. PubMed Abstract | Publisher Full Text\n\nhttp\n\nKull M Jr, Kallikorm R, Tamm A, et al.: Seasonal variance of 25-(OH) vitamin D in the general population of Estonia, a Northern European country. BMC Public Health. 2009; 9: 22. PubMed Abstract | Publisher Full Text\n\nHwalla N, Al Dhaheri AS, Radwan H, et al.: The prevalence of micronutrient deficiencies and inadequacies in the Middle East and approaches to interventions. Nutrients. 2017; 9: 229.\n\nWang TJ, Zhang F, Richards JB, et al.: Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet. 2010; 376: 180â188. PubMed Abstract | Publisher Full Text\n\nHeaney RP: Vitamin D and calcium interactions: functional outcomes. Am. J. Clin. Nutr. 2008; 88: 541Sâ544S. PubMed Abstract | Publisher Full Text\n\nSharafi N, Fatima A, Gillani SW, et al.:Parents reported Vitamin D Supplementation among Children (Responses). figshare. Dataset.2022. Publisher Full Text"
}
|
[
{
"id": "157818",
"date": "14 Dec 2022",
"name": "Tauqeer Hussain Mallhi",
"expertise": [
"Reviewer Expertise Pharmacy Practice",
"Clinical Pharmacy",
"Pharmacotherapy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the invitation to review this study. The authors made a good effort to explore the use of vitamin D supplementation among children in UAE. I have a few comments on this manuscript.\nThe sample size estimation can be omitted from the abstract section.\n\nPlease provide a few references that aid in the development of data collection form.\n\nPlease provide information on the validation and reliability of the data collection form.\n\nPlease clarify that the data was collected through interviews or self-administration techniques.\n\nThe quality of figure 1 can be improved.\n\nConvenient sampling is also the limitation of this study that precludes the generatability of the findings.\n\nThere is a need to provide some information on the barriers of vitamin d supplementation in children.\n\nThe manuscript will require some corrections in syntax at some places.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9379",
"date": "13 Mar 2023",
"name": "Syed Wasif Gillani",
"role": "Author Response",
"response": "Comment 1: The sample size estimation can be omitted from the abstract section. Response 1: we agree with this comment therefore; we have accordingly omitted the sample size estimation from abstract of the manuscript. Comment 2: Please provide a few references that aid in the development of data collection form. Response 2: Thank you for this suggestion. The references list is updated and the new references are incorporated in the list to emphasize this point. Comment 3: Please provide information on the validation and reliability of the data collection form. Response 3: Thank you for pointing this out. We have added the suggested content to the manuscript. Comment 4: Please clarify that the data was collected through interviews or self-administration techniques. Response 4: We agree with the reviewerâs assessment. Accordingly, throughout the manuscript, we have clarified that the data was collected on a questionnaire which was filled by the investigators. The investigator verbally inquired questions to the included parents individually from the questionnaire and filled the data on their behalves as they answered Comment 5: The quality of figure 1 can be improved. Response 5: As suggested by the reviewer, we have updated figure 1 with improved quality in the results section of the manuscript. Comment 6: Convenient sampling is also the limitation of this study that precludes the generatability of the findings. Response 6: We agree that this is a potential limitation of the study. We have added this as a limitation in the discussion part of the manuscript. Comment 7: There is a need to provide some information on the barriers of vitamin d supplementation in children. Response 7: We agree with this and have incorporated your suggestion throughout the manuscript."
}
]
},
{
"id": "157815",
"date": "19 Dec 2022",
"name": "Muhammad Umair Khan",
"expertise": [
"Reviewer Expertise Medicine optimisation",
"mental health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study explores an important subject on vitamin D supplementation among children in the United Arab Emirates. However, the manuscript needs significant revision before it can be published. I have raised some questions/comments in each section which authors may consider in preparing their revisions.\n\nAbstract\nBackground\nI suggest using âinvestigateâ or âassessâ rather than review. Authors can avoid âgeneral publicâ. âamong childrenâ is just fine.\n\nMethods\nConsider adding information like: who were the participants? Where was the study conducted? Any statistics used?\n\nResults\nAs per the information provided, 89.1% were mothers and 15.7% were fathers. It gives a total of 104.8%. Please check these numbers again.\n\nConclusion\nPlease reconsider the conclusion. It is not clear what the authors are trying to suggest.\n\nIntroduction\n1st paragraph â last sentence â for the ease of readers, please explain what happens when 25(OH) D transforms to 1,25 (OH) D.\n\n3rd paragraph â last sentence - 5000 units daily in patients over 12 months old â is there any upper age limit and it applies to all including adults?\n\n4th paragraph - What about epidemiological studies in children? Are there any? What do they say? In my opinion, it is important to answer these questions as the population of interest in this is children.\n\n7th paragraph â first sentence â in children, adults, or general population?\n\n8th paragraph â first sentence â too long to comprehend. Please rewrite to improve clarification.\n\n8th paragraph â second sentence - What is the prevalence of vitamin D deficiency among adults and children globally and in the UAE?\n\n8th paragraph â second sentence - Are authors trying to imply that RCTs are the most appropriate research designs to determine prevalence? Please reconsider and rewrite this sentence.\n\n9th paragraph â I would suggest write a broader aim and then the objectives that this study tried to achieve.\n\nAlso, the phrase âamong children in the general publicâ sounds a bit odd to me. I think âamong childrenâ in just fine.\n\nMethods\nCan authors provide some examples of the public places that were used?\n\nCan authors provide some references from where the questionnaire was adapted? Where were those studies conducted? Are there any cultural differences? How did the authors account for that?\n\nHow many questions were there in total and in each section?\n\nWhich section had yes/no and multiple response questions? Were there mixed question in each section?\n\nHow did the authors evaluate the barriers to supplementation? The three sections do not tell anything about the barriers.\n\nHow did the authors investigate the reasons for vitamin D deficiency? I am not sure it was mentioned earlier until this point.\n\nI am not sure what the authors are trying to tell through the primary outcomes. The first two points look like the objectives. The third point looks like a hypothesis, but I am not sure what that means.\n\nAround 319 participants were expected â Are the authors trying to imply that 319 participants were contacted?\n\nResponse rate is excellent. How do the authors compare this with the similar other studies?\n\nMissing data â surprising!\n\nI suggest authors should mention earlier in the methods section that parents completed the survey.\n\nWhether the questionnaire was self-administered or interviewer-administered?\n\nLooking at the information presented in âconsent formâ, it appears that the questionnaire was self-administered. Did the parents completed the questionnaire right there and then? How did they return the questionnaire?\n\nWhy the authors excluded children with minor illnesses as well as those with cognitive impairment? It was parents who completed the survey.\n\nA chi-square analysis was done to âŠâŠâŠ..?\n\nBoth a tabular and graphic version of the data was used to show it â generally we use one form of representation to avoid duplication.\n\nBias â Given this is an observational study, how can the authors assure this?\n\nAuthors did not mention about the language in which the questionnaire was designed and distributed to the participants.\n\nResults\n1st sentence â It was mentioned earlier. Avoid duplication.\n\n2nd sentence â I think the brackets are not utilised appropriately.\n\nFrom what I understand, 203 mothers and 39 fathers participated in the study. It makes a total of 242 participants. What about other 6 participants?\n\nIf 203 is the correct number, it is 81.8% of the total participants not 89.1%.\n\nAs per the information provided, 89.1% were mothers and 15.7% were fathers. It gives a total of 104.8%. Please check these numbers again.\n\nI strongly suggest authors to review their calculations and rewrite the results section.\n\nThe name of countries in Fig 1 was not readable.\n\nIt was mentioned earlier that chi-square test was used but the results section did not mention chi-square values and any significance values.\n\nDiscussion\nThe first 3 paragraphs of the discussion are simply results. I suggest authors to summarise the key results in the first paragraph to avoid duplication.\n\nI think the discussion section needs considerable revision. Currently, it seems like a results section. I suggest authors to focus more explaining the results in light of the broader literature on the topic.\n\nThe results of this study are generalizable â this statement needs more clarification. Earlier authors discussed the climate issues is UAE which is a contributing factor. Is the climate same in different parts of the world? Europe/US/Australia/Africa? Is not, how will the authors support their statement on generalisability?\n\nConclusion\nI suggest authors to reconsider their conclusion. I am not sure what authors are trying to imply by saying that financial background of parents can aid in addressing the challenges of vitamin D supplementation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9380",
"date": "13 Mar 2023",
"name": "Syed Wasif Gillani",
"role": "Author Response",
"response": "Comment 1: Background I suggest using âinvestigateâ or âassessâ rather than review. Authors can avoid âgeneral publicâ. âamong childrenâ is just fine Response 1: We agree with this comment therefore we have changed the word âreviewâ to âassessâ. We also agree to omit âgeneral publicâ and mention âamong childrenâ. Comment 2: Methods Consider adding information like: who were the participants? Where was the study conducted? Any statistics used? Response 2: Thank you for this suggestion. We have added better information for the method section which is now clearer. We have clarified the survey was conducted in public places like malls, parks and hospitals in the U.A.E and the study participants were parents/caregivers of children from ages 4-to 15- years. The questionnaire was both self-administered and interviewer-administered. For parents/care givers who found it difficult to understand the questionnaire, the interviewer verbally inquired questions to the included participants individually from the questionnaire and filled the data on their behalves as they answered. The questionnaire was filled on a secure device with only the main investigators having access to it. Comment 3: Results As per the information provided, 89.1% were mothers and 15.7% were fathers. It gives a total of 104.8%. Please check these numbers again. Response 3: We agree with the reviewerâs comment. We have recalculated our values and changed the numbers to the right ones.  Comment 4: Conclusion Please reconsider the conclusion. It is not clear what the authors are trying to suggest. Response 4: As suggested by the reviewer we have rephrased the conclusion to clearer one in the manuscript. Comment 5: 1 paragraph â last sentence â for the ease of readers, please explain what happens when 25(OH) D transforms to 1,25 (OH) D Response 5: We agree to the reviewerâs comment and we have added information for the reader to understand what happens when 25(OH) D transforms to 1,25 (OH) D Comment 6 3 paragraph â last sentence - 5000 units daily in patients over 12 months old â is there any upper age limit and it applies to all including adults? Response 6: We agree with this suggestion from the reviewer. Therefore, we have mentioned that for the treatment of vitamin D deficiency rickets, the AAP recommends an initial 2- to 3-month regimen of âhigh-doseâ vitamin D therapy of 1000 units daily in neonates, 1000 to 5000 units daily in infants 1 to 12 months old, and 5000 units daily in patients over 12 months old. Comment 7 4 paragraph - What about epidemiological studies in children? Are there any? What do they say? In my opinion, it is important to answer these questions as the population of interest in this is children. Response 7: Thank you for this suggestion. We agree to add some information on epidemiological studies in children. Therefore, we have mentioned that it is estimated that the prevalence of deficiency is 62â95.7% in new-borns and breast-feeding groups (0â6 months), 46â80% in 6â60 months of age and 37.8â97.5% in 5â20-year-old children. Comment 8 7 paragraph â 4rst sentence â in children, adults, or general population? Response 8: We have now added âgeneral populationâ in the paragraph. Comment 9 8 paragraph â 4rst sentence â too long to comprehend. Please rewrite to improve clari4cation. Response 9: We thank the reviewer for his comment, we have paraphrased the sentence to a shorter and clearer one. Comment 10 8 paragraph â second sentence - What is the prevalence of vitamin D de4ciency among adults and children globally and in the UAE? Response 10: We have answered this comment from the reviewer in the 4th paragraph of our introduction. Comment 11 9 paragraph â I would suggest write a broader aim and then the objectives that this study tried to achieve Response: We thank the reviewer for his suggestion here. We have now mentioned our aim, the need of this study to be conducted, and the high prevalence of vitamin D deficiency in the above paragraphs in a better pattern. Comment 12 Also, the phrase âamong children in the general publicâ sounds a bit odd to me. I think âamong childrenâ in just one. Response: We agree with the reviewer and have omitted the phrase âin the general publicâ Comment 13 Methods Can authors provide some examples of the public places that were used? Response: Yes, we have provided some examples of the public places that were used in our study. Comment 14 Can authors provide some references from where the questionnaire was adapted? Where were those studies conducted? Are there any cultural differences? How did the authors account for that? Response: Yes, we have provided the reference for the other study that we adapted the questionnaire from. We have also mentioned where the other study was conducted. However, our study has no association with the other studyâs results or population. We have only adapted some of the questions from that studyâs questionnaire. Comment 15 How many questions were there in total and in each section? Response: Thanking the reviewer for this keen observation. We have now mentioned the total number of questions in our questionnaire and the number of sections as well. Comment 16 Which section had yes/no and multiple response questions? Were there mixed question in each section? Response: Sections apart from the demographics, consisted of multiple responses and yes/no questions. Yes there were mixed sort of questions in some sections too. Comment 17 How did the authors evaluate the barriers to supplementation? The three sections do not tell anything about the barriers. Response: We thank the reviewer for his very thoughtful suggestion. It was important to mention about the barriers the parents face with providing VD supplementation to their children. Comment 18 How did the authors investigate the reasons for vitamin D de4ciency? I am not sure it was mentioned earlier until this point. Response: Thank you for this suggestion. We have rephrased the sentence and mentioned how we investigated the reasons for VDD. Comment 19 I am not sure what the authors are trying to tell through the primary outcomes. The 4rst two points look like the objectives. The third point looks like a hypothesis, but I am not sure what that means. Response: Thank you for this comment, we have paraphrased the primary outcomes in the manuscript. Comment 20 Around 319 participants were expected â Are the authors trying to imply that 319 participants were contacted? Response: Thank you for this suggestion. We have rephrased the sentence to make better understanding for the reader on the number of study participants determined by Rao soft sample size calculator and the number of participants who responded and actively participated in our study. Comment 21 Response rate is excellent. How do the authors compare this with the similar other studies? Response: Thank you for this comment, association between the response rate and the sample size is such that the response rate increases when the sample size less than 300 participants. Comment 22 Missing data â surprising! Response: We have rephrased this to A total of 248 participants were included and all 248 of them actively participated in our study. During analysis, we found no missing data from our participants. Comment 23 ⢠I suggest authors should mention earlier in the methods section that parents completed the survey. Response: Thank you for suggesting, we have done this now. Comment 24 ⢠Whether the questionnaire was self-administered or interviewer-administered Response: Mentioned now in the manuscript that it was interviewer administered. Thank you. Comment 25 Looking at the information presented in âconsent formâ, it appears that the questionnaire was self-administered. Did the parents completed the questionnaire right there and then? How did they return the questionnaire? Response: The questionnaire was both self-administered and interviewer-administered. The questionnaire content was described before letting the participants administer the data into it and the written consent form was taken from each participant prior to interviewing/ handing out the questionnaire. Comment 26 Why the authors excluded children with minor illnesses as well as those with cognitive impairment? It was parents who completed the survey Response: The data was filled by the parents/ care givers but it was about children. Therefore, to avoid bias between the healthy children and ill children we excluded children with minor illnesses and those with cognitive impairment. Comment 27 Both a tabular and graphic version of the data was used to show it â generally we use one form of representation to avoid duplication Response: Thank you for this suggestion but we have not used both the tabular and graphic version for one same piece of data. The frequency of nationality bar graph is different than that of tabular form, where the nationality is associated with supplementation practice. Comment 28 Bias â Given this is an observational study, how can the authors assure this Response: Thank you for this suggestion. We have clarified our bias now. Comment 28 Authors did not mention about the language in which the questionnaire was designed and distributed to the participants Response: Thank you, we have now mentioned that the article was generated in both English and Arabic Comment 29 Results 1 sentence â It was mentioned earlier. Avoid duplication. Response: Thank you. Duplication undone. Comment 30 2 sentence â I think the brackets are not utilised appropriately. Response: Thank you for this comment, we have looked into it. Comment 31 From what I understand, 203 mothers and 39 fathers participated in the study. It makes a total of 242 participants. What about other 6 participants? Response: Thank you for this correction. We have recalculated and put in the right figures. Comment 32 ⢠If 203 is the correct number, it is 81.8% of the total participants not 89.1%. Response: The calculation and figures are now corrected. We have put the right percentage now. Thank you. Comment 33 As per the information provided, 89.1% were mothers and 15.7% were fathers. It gives a total of 104.8%. Please check these numbers again Response: Thank you. The correction for percentages of mothers and fathers in our study has now been corrected. Comment 34 ⢠I strongly suggest authors to review their calculations and rewrite the results section.   Response: Thank you for this suggestion, we appreciate your concern. We have reanalyzed our numbers and percentages and now have put in the right numbers. We have re-written our results. Comment 35 The name of countries in Fig 1 was not readable Response: Thank you, we have added a readable picture. Comment 36 It was mentioned earlier that chi-square test was used but the results section did not mention chi-square values and any signi4cance values. Response: the result part has been revised accrodingly Comment 37 The 4rst 3 paragraphs of the discussion are simply results. I suggest authors to summarise the key results in the 4rst paragraph to avoid duplication. Response: Thank you for your highly appreciated suggestion. We have summarized the discussion part to a concise and clear paragraph now. Comment 38 ⢠I think the discussion section needs considerable revision. Currently, it seems like a results section. I suggest authors to focus more explaining the results in light of the broader literature on the topic Response: Yes, we agree with the reviewerâs comment. Therefore, we have significantly made improvement in this particular section of our manuscript. Comment 39 The results of this study are generalizable â this statement needs more clari4cation. Earlier authors discussed the climate issues is UAE which is a contributing factor. Is the climate same in different parts of the world? Europe/US/Australia/Africa? Is not, how will the authors support their statement on generalisability?   Response: Yes, we appreciate and agree with this comment. We have changed the phrase to- The results of this study are generalizable since it has been conducted on general population of children from multiple ethnicities in the U.A.E. Comment 40 Conclusion  I suggest authors to reconsider their conclusion. I am not sure what authors areâŠ.  Response: Thank you so much for pointing out this suggestion to us. We have rephrased our conclusion to a very clear cut, understandable one now"
}
]
}
] | 1
|
https://f1000research.com/articles/11-1456
|
https://f1000research.com/articles/12-265/v1
|
13 Mar 23
|
{
"type": "Case Study",
"title": "Smart capital cities: towards a smart new administrative capital (NAC)",
"authors": [
"Adel Samy El menchawy",
"Hamad Hassan Moustafa",
"Nada Ibrahim Abdel-Hamid",
"Adel Samy El menchawy",
"Hamad Hassan Moustafa"
],
"abstract": "Overpopulation and resource shortages are becoming increasingly important challenges to our global agenda. Cities are rapidly evolving, expanding in size and density while confronting major challenges such as migration, deteriorating healthcare, and transportation issues. Studying Egypt's overall urban development tendencies, as well as the continuously growing population in Greater Cairo, it becomes clear that developing new urban communities to accommodate this increase is a requirement. The major goal of any city pursuing evolution is to solve its issues to improve the quality of life and services for its residents, which ties into the goal of a \"smart city,\" as it maximizes the use of technology to satisfy the current and the future needs. This paper discusses the strategy for transforming the old Cairo capital into a new administrative capital (NAC), which is intended to be a smart capital city. It also discusses smart city dimensions, smart city performance evaluation criteria, and smart city technologies such as implementing smart applications in various city sectors, all of which contribute to the city's continued development.",
"keywords": [
"New Administrative Capital",
"Over-population",
"Smart city",
"Smart Applications",
"Smart Technology"
],
"content": "Introduction\n\nA smart capital city is one in which smart technologies are used in almost every aspect of daily life, forming a connected technological network that provides services and solves capital city-related issues.\n\nThe idea of establishing a new administrative center outside of Greater Cairo is neither new nor unique in Egypt. âThe issue of relocating Egyptâs capital away from Cairo has periodically surfaced in semi-serious national discussion over the last twenty years,â as noted in 1994.1\n\nSince the sudden migration of citizens from rural areas to urban settlements in the late 1950s, Cairoâs administrators have been struggling with the cityâs rapidly growing population and rapid urbanization as a number of significant issues have emerged, including the rise in urbanism rates, unplanned urban growth, and low living standards, due to the steady population increase and consumption of limited energy resources and currently available resources. Thatâs why cities have lost their environmental balance, as overpopulation has severe environmental implications and is followed by number of accumulative problems including a lack of affordable housing, urban sprawl and land consumption, transportation problems, health problems, poor air and water quality, energy inefficiency and communication problems.2\n\nCapital cities are perceived as places where one could have a better life, because of better opportunities, higher salaries, better services, and better lifestyles. Thus, using smart technologies in new capital cities could also help in solving problems, due to the existence of smart applications in various fields throughout the city.\n\nThis paperâs main objective is to ensure that smart cities make services in the city more efficient and enhance its competitive capabilities while ensuring that it meets the needs of current and future generations in terms of economic, social, and cultural aspects. It also discusses successful examples of smart cities which succeeded in improving their circumstances after implementing smart technologies in their different life aspects, which aims to highlight the importance of smart applications and their role and effectiveness in the success of smart cities, in addition to clarifying the importance of smart cities in ensuring a better life for its citizens with fewer challenges to face.\n\n\nBackground\n\nSince their first appearance in the literature in 1997, smart cities have exploded in popularity, attracting major scientific and industrial attention. The goal of smart communities is to promote cities that provide basic infrastructure and provide a fair quality of life for their residents, as well as a clean and sustainable environment using âsmart applicationâ solutions.\n\nThe smart cityâs dimensions are linked to traditional urban development and growth theories like transportation, economy, natural resources, quality of life, and participation. Figure 1 shows these six dimensions that are involved in the use of information and communications technology (ICT) and innovation by cities (new, existing, or districts) to maintain economic, social, and environmental sustainability and address multiple challenges (people, economy, governance, mobility, environment and living).3\n\nA simple hierarchic structure shown in Figure 2 was developed to describe a smart city and its six dimensions, where each level is described by the results of the level below. As a result, each dimension is defined by a set of factors and each of which is described by a set of indicators.\n\nThe factors were defined in several workshops, with the overall goal of smart city development in mind. Finally, 33 factors were chosen to describe the six dimensions. Those six dimensions and their assigned factors represented in Figure 3 below help researchers in developing a framework for evaluating the performance of smart cities. Innovation, entrepreneurship, trademarks, productivity, and labor market flexibility, as well as integration in the (inter-)national market, are all factors that contribute to a smart economy. Smart people are defined not only by their level of qualification or education, but also by the quality of their social interactions in terms of integration and public life, as well as their openness to the âoutsideâ world. Political involvement, citizen services, and administrative efficiency all fall under the umbrella of smart governance.4\n\nAll Figures 1, 2 & 3 were acquired from Rudolf Giffinger journal article with a permission and were edited by the researcher.\n\n\nGlobal classifications (frameworks and models used to measure the performance of smart cities)\n\nThe evaluation of smart city performance is a useful tool for future development. Citiesâ strengths and weaknesses can be identified through this evaluation, allowing the formulation of development goals and strategies. This assessment is required for:\n\n⢠Comparing cities and obtaining the best experience.\n\n⢠Recognizing weaknesses and calculating the amount of effort required to overcome them.\n\n⢠Assessing the current state of development in the city in comparison to other cities, as well as identifying potential development opportunities.\n\n⢠The potential to draw individual attention to smart city development issues.\n\nMany models exist to assess the performance of smart cities, though they may differ in terms of measurement indicators to which a relative weight is assigned for quantitative evaluation. These framework models are:\n\nThe City Keys framework has five basic dimensions, with relative weights ranging from 13% to 27%, and weights distributed to each dimension based on performance measurement indicators.\n\nA set of indicators was designed to evaluate smart city projects and the performance of smart cities for the City Keys framework based on a study of indicators from 43 existing indicators; new indicators were also proposed to fill gaps in existing frameworks, mostly related to specific characteristics of smart city projects.\n\nThe following is how the indicators were organized in a framework: people, planet, and prosperity, with specific smart city indicators and sub-themes that match key policy goals, were identified. Indicators for evaluating smart city projects that assess or evaluate individual projects, indicate the impact of the project, or compare projects. City indicators can be used to show the extent to which public policy objectives have been met. Smart city indicators focus on monitoring the development of the city towards a smarter city development over time.5\n\nThe ISO/TS 37151 standard was published in October 2015 and covers:\n\n⢠Smart community infrastructures\n\n⢠Principles and requirements for performance metrics\n\nISO is a global federation of national standards organizations (ISO member bodies). International standards are usually developed by ISO technical commissions, and each interested member body has the right to form a technical committee and be represented on it. Intergovernmental and nongovernmental organizations also participate in collaboration with the (ISO) technical standardization committee.\n\nISO/TC 268 on sustainable development in communities and SC1 on smart community infrastructures are the committees responsible for this document. Quality of life, economic growth, poverty reduction, pollution control, and congestion relief are all goals that communities strive to achieve. Communities and their activities benefit from community infrastructure such as energy, water, transportation, waste management, and ICT.\n\nAs a result, investing in such infrastructures allows communities to achieve internationally recognized societal goals (such as the Millennium Development Goals of the United Nations [MDGs]).5\n\nA group of researchers from the Technical University of Viennaâs Regional Science Center identified 31 factors to evaluate smart cities, which fall under the smart cityâs dimensions (smart economy, smart people, smart government, smart mobility, smart environment, smart life), and selected 74 indicators to analyze the performance of each factor.\n\nThe criteria were based on the smart city concept, which was used to express areas that use information and communication technologies, cities with smart industries, industries that use technologies in production processes, as well as cities that seek to develop the education system, in other words, the city with the most intelligent people.\n\nThe interaction between the government and citizens, as well as the employment of new technologies in daily life, are all part of this concept. Technology refers to a variety of ideas such as modern transportation technologies, sustainability, energy, security, and safety, in addition to information and communication technologies. Each level of the assessment expresses the level before it, so each dimension is represented by several factors, and each factor is represented by indicators in Table 1.5\n\nThe relative weight is evenly distributed among the six main dimensions, with each accounting for 16.67 percent of the overall relative weight. The relative weight of each sub-criterion is shown separately in Table 1. According to 2015 data, the Vienna Technical University framework (as an integrated framework) was used to assess the performance of seven European cities: Bonn, Bristol, Stockholm, Rennes, Helsinki, Amsterdam, and Copenhagen. As illustrated in Figure 4 and Figure 5, the six dimensions of the framework are rated on a scale of +2 to -2 for each dimension; the last column shows the overall average rating. Figure 5 illustrates the composition of the total rating by characteristics for each city.\n\nFigures 4 & 5 were acquired from Rudolf Giffinger journal article with permission and were edited by the researcher.\n\nA smart capital city, which incorporates information and communication technologies (ICT) to improve the quality and performance of urban services such as energy, transportation, and utilities, and uses smart application systems to cover the dimensions of the smart city in order to reduce resource consumption, wastage, overall costs, and to improve the quality of living for its citizens through smart technology, could be a solution given to a deteriorated overcrowded capital city. Studying successful examples that have faced comparable issues can facilitate the development of one of these evaluation criteria, resulting in a framework that can be used to assess the performance of smart cities.3\n\n\nRelated work\n\nIn 1999, Cyberjaya was the first Malaysian smart city to launch. Its main goal was to serve as a hub for information technology (IT) companies, encouraging investors to set up shop there.\n\nThe city used a variety of applications, including:\n\n- E-government: The concept of e-government is used in administrative centers to improve communication between citizens, businesses, and government agencies by utilizing information and communication technology.\n\n- Multi-function card: A smart card called âMyKadâ is used as an identity card, credit, ATM card, driverâs license, and health card.\n\n- Smart schools: Internet and ICTs are provided to schools, allowing students to participate in virtual lessons and obtain knowledge through technology.\n\n- Development and research center: Conducting research on ICTs and development is one of the goals of the multimedia corridor project, which encourages academic and scientific institutions to collaborate on research.\n\n- Telehealth: This project use information and communication technology to access health care services remotely and manage human health care to deliver telehealth services as the multimedia corridor initiative aims to establish a regional telehealth center.\n\n- E-Business: Attract local and foreign businesses to work on the Internet and in multimedia industries.6\n\nThe smart city applications developed by Cyberjaya aim to support the multimedia corridor projectâs growth, increase competitiveness, and close the digital divide, which is the gap between demographics and regions with access to modern information and communication technology such as telephones, televisions, computers, and the internet.\n\nSongdo was built as a growth pole in the multi-central region of the capital city. It is a financial, technological, and research center that focuses on technical and knowledge-based sectors as the primary engines of Korean economic growth.\n\nThis city is known as the âNortheast Asia Gatewayâ and is known as the International City or Smart City. Songdo followed a sustainable development strategy to solve environmental challenges, it is ranked 122 out of 146 in the 2005 Sustainable Environmental Classification, while Korea was classified among the top 10 countries in CO2 emissions by the International Energy Agency in 2005.\n\nSongdo used a variety of applications, including:\n\n- Songdo Techno-Park: A 45-hectare industrial complex that was built between 1998 and 2005. It has five main divisions: the main management center, the biotechnology trade research and development center, the Korea Institute of Industrial Technology, the Incheon University Research Center, and the Inha University Research Center, as well as 35 business and research and development centers. Its goal is to create a conducive and supportive environment for businesses to develop and expand their capacity and production to benefit Koreaâs national economy.7\n\n- Smart environment: The city has committed to the LEED Green Building Classification System through several strategies, including reducing carbon emissions, using environmentally friendly transit systems, and providing green spaces and places. This city is a âpedestrian city,â with open spaces, pedestrian pathways, and public gathering spots connecting all residential structures.\n\n- Providing services through using network: The computing environment allows residents and visitors to access a variety of services from anywhere, at any time, in a variety of regions, thereby improving their quality of life.\n\n- Disaster Management System.\n\n- System for security and safety.\n\n- E-learning.\n\n- Telehealth.\n\n- Automatic traffic control.8\n\nSongdo provides good communications, transport and cultural facilities, infrastructure, business climate and better quality of life, and offers incentives for foreign investment and multinational companies through tax exemptions for foreign investment.\n\nThe transformation of Dubai into a smart city is part of the United Arab Emirates (UAE) governmentâs aim to strengthen Dubaiâs commercial and economic hub, rather than relying solely on the oil economy. The objectives were to use information and communication technology to create an atmosphere that draws foreign corporations, so promoting the local economy, as well as to attract clever talent, as the knowledge economy is dependent on humans.\n\nDubaiâs smart applications are:\n\n- The electronic government: Transformation of government to e-government means that government departments may provide the greatest, most effective, and efficient public services for both citizens and businesses by reformulating the numerous services supplied by all departments using the internet and other electronic technologies.4\n\n- E-Commerce: E-commerce has a significant impact on the local and worldwide business environment, contributing to the radical transformation of economic activity and the social environment. The main goal is to turn Dubai into a significant regional online business hub, sparking regional rivalry for the establishment of new cyber cities. Dubai launched a business e-market (Tejari.com), a corporate electronic gateway, in the year 2000, to provide a variety of commercial items to the region.\n\n- E-Learning: E-learning is a significant component of smart cities since it merges information and communication technology with education. Its goal is to promote e-culture throughout the community. To prepare a generation capable of exploiting technology, computer-related materials have been introduced in schools. School computers were also outfitted with cameras, allowing parents to monitor their children at school from the comfort of their own homes.4\n\n- Intelligent transportation: Modern technologies such as parking management and control systems, traffic signal management systems, and emergency traffic management systems all strive to improve the efficiency of the road network\n\n- Dubai Technology, E-Commerce, and Media Free Zone: Located in the Jebel Ali Free Zone, it is a corporate organization with financial and administrative independence.\n\n- Dubai Oasis: A state-of-the-art service area that provides the highest levels of luxury, and it serves as a model for utilizing the latest technology and capabilities supplied by the Dubai Free Zone infrastructure.\n\n- Electronic Banks: Electronic banks use the Internet to deliver financial services, and e-commerce is the primary driver behind the growth of electronic banks.\n\n- Dubai Internet City (DIC): DIC was established as an important technological base, providing facilities and infrastructure that attract international technology companies to be localized and integrated in local and regional markets, thus providing digital services to more people, faster, and aims to attract new ideas in the world of the Internet by providing an environment that embraces distinct ideas and projects.9\n\nDubai has an electronic administration center that communicates with the public using electronic and multi-media channels, putting the city on the worldwide map. Apart from that, the government should promote the telecommunications industry and infrastructure by creating an investment climate that attracts investment, experience, and well-known international corporations, as well as reducing barriers for foreign investors in computer software and hardware. As concluded from studying the previous Asian examples; It was clarified that new smart cities need few elements to exist, these requirements were repeated in most of the previous examples which are ICT infrastructure, building skills and abilities, and âsmart applicationsâ which were distinguished to be used to bring intelligence to cities. Each application refers to a certain dimension from the six dimensions of the smart city.\n\nThe use of ICTs to improve and develop working in many sectors of the smart city has been proven in a number of nations. As a result, it is critical to develop electronic applications in critical sectors as shown in Table 2, and these apps should be adapted to the preferences of each city, whether in the fields of education, health, government, tourism, or industry. To get the most out of electronic applications, they should be adapted to the local needs, available to the broadest possible audience, and simple to use.\n\nBristol is geographically well-connected to the UK, Europe, and the rest of the world. The port and airport provide important links for business to international partners. Bristol Airport is the UKâs ninth largest airport, and the fifth largest outside of London, with ambitions to rapidly expand to 12 million passengers per year.\n\nThe following are some of the problems that Bristol faces:\n\n- The need for a long-term response to climate change and urbanization. The council unanimously agreed in November 2018 to set a goal for the city to be carbon neutral by 2030.\n\n- Affordability of housing â the cheapest Bristol property costs more than eight times the annual incomes of the poorest households, resulting in many individuals sleeping rough or homeless.\n\n- One of the UKâs most congested cities, with 300 deaths attributed to air pollution each year.10\n\nSmartness has the following characteristics:\n\n1- Smart City Bristol, which focuses on smart energy, smart transportation, and smart data, was founded in 2011. Bristol prioritizes reducing car use to minimize greenhouse gas emissions; thus, a disused railway has been transformed into a traffic-free route and a safe zone for cyclists.\n\n2- Smart motorways â In England, âsmart motorwaysâ have been implemented, which use innovative technologies to actively control traffic flows and improve journey times, such as variable speed restrictions.\n\n3- Smart safety â Collision Recording and Sharing (CRASH), a new road event recording system that captures accidents and classifies injuries, has provided England with enhanced accident data. This has the potential to help prevent accidents.\n\n4- Bristol Operations Centre â A smart city center that keeps the city moving while also keeping it safe and healthy. It is a single center that provides traffic management, bus lane enforcement, public space CCTV surveillance, call processing, and security alarm monitoring, among other services.\n\n5- Bristol City Council Information Technology (IT) Services â ensures and governs all aspects of IT delivery and supporting strategy across the council.\n\n6- Bristol wants to cut carbon dioxide emissions by 40% by 2020 and 80% by 2050 and has developed a portable gadget called the âAir Quality Monitoring Deviceâ that gathers air composition to estimate air quality. Bristol Energy is the countryâs first energy firm to supply 100 percent green electricity and reinvest its profits back into the community (Intelligent environment).11\n\nStockholm was named as the first European Green Capital in 2010. The assessment was based on a number of criteria, including climate impact, local transports, green areas and air quality.\n\nOne of the issues facing Stockholm is that the population is predicted to grow by more than 11% between 2014 and 2020, or 50 people each day on average. Furthermore, Sweden is one of the worldâs top ten most globalised countries, resulting in increased international travel and communication to the capital.12\n\nThe City of Stockholm approved the Smart and Connected City Strategy in 2017, which claims that by 2040, the city intends to be the worldâs smartest and most connected city.\n\nFeatures of being smart:\n\nThe capital has the worldâs largest open fiber network and a 100 percent broadband coverage12 which helps in:\n\n1- Reducing greenhouse gas emissions maximum 1.5 tones CO2 per resident aiming for a fossil-free and climate-positive Stockholm by 2040 (smart environment).\n\n2- Smart waste handling turning waste to electricity, heat, and biogas for vehicles.\n\n3- Smart traffic management, Traffic control: Main bus lines are prioritized at traffic lights before other sorts of traffic.\n\n4- Smart lighting: Smart lighting is a component of the Grow Smarter project, which involves a collaboration between eight European cities and several green-tech companies to test and develop smart solutions for urban sustainable growth, such as (sensor-controlled LED lighting) for pedestrian and bicycle paths. Motion-activated LED lighting is controlled by a customized mesh network of radio transmitters embedded in lampposts.\n\nHelsinki, the capital of Finland, is the administrative center of the country. With a population of 650,000, the total area of the city is 719 km2. One of the problems Helsinki faces is the rapid growth in the population, as it is growing at a rate of 0.75-1.2%, which increases the need for smart management of city resources.13\n\nFeatures of being smart: improving the personnelâs capabilities in emerging digital technologies, such as artificial intelligence (AI), robotics and a new data-based concept of âsmart educationâ, is one of Helsinkiâs aims. The smart education concept utilizes data analytics, providing more individual learning designs and experiences.14\n\n1- The Economic Development and Planning Division is the head of the ICT and data administration department of Helsinki city, and is responsible for the development of digital technologies in diverse city domains.\n\n2- Smart mobility: Helsinki offers remarkable opportunities in developing smart mobility ecosystem, which includes innovative apps to electric cars, automated vehicles, and driverless busses. This is all due to the clean tech, electric vehicle specialists and technicians.\n\n3- Shifting Transportation Mode Share: riding or walking to work are initiatives for several governments, and are gradually gaining momentum which makes the city smarter and the environment friendlier.14\n\nHouseholdsâ proportion with more than two working adults were 23.1%, 67.0%, and 73.3%, respectively.\n\nHelsinki City Council helps to encourage innovation by offering a platform for bringing together various smart city stakeholders, including application developers, investors, and regulatory organizations.\n\nSo, As concluded from studying the previous European examples, it is clarified that Smart applications vary from one smart city to another, but they all contribute to improving the quality of smart city dimensionsâ factors by improving city services and living experiences of citizens as shown in Tables 3, 4 & 5. To measure the performance of smart cities, Vienna University technical framework could be used which has been applied to seven European cities in 2015 by rating is in the range from 2 to -2\n\nSmart applications vary each city, depending on citizen expectations; they help to achieve the smart city dimensionâs goal to improve city services. As shown in Tables 6 & 7, for the previous studied European examples examined in 2015, the dimension in which each city focused on building its related smart apps had a higher value than the other dimensions. This shows that smart application has an impact on the value of smart dimension evaluations, which in turn has an impact on the overall smartness of a city.\n\n\n\n1. A device called âAir Quality Monitoring Deviceâ that collects air composition in order to determine the air quality is used aiming to reduce carbon dioxide emissions by 40%\n\n2. Bristol City Council (IT) services\n\n\n\n1. Smart waste handling turning waste to electricity\n\n2. Smart traffic and lighting reduce waiting times lead to reduced environmental impact\n\n3. Reducing greenhouse gas emissions maximum 1.5 tones CO2 per resident\n\n\n\n1. Improve its personnelâs capabilities in emerging digital technologies, such as AI and robotics\n\n2. âSmart educationâ is set to provide more individual learning design and experiences\n\n3. The ICT and data administration department of Helsinki city operates under its Economic Development to improve digital technologies in diverse city domains\n\n\nThe new administrative capital (NAC, the proposed framework)\n\nThe idea of moving the capital is neither new nor an experiment, but rather a process that most countries in the globe have gone through in ancient and modern history, the first of which is Egypt, which has relocated its capital 17 times throughout its history.15\n\nHowever, after the overpopulation crisis in Cairo and by the increasingly swelling number of residents and the continuous shrinking number of resources, Presidential Decree No. 57 of 2016 was issued, giving area south of Cairo Suez shown in Figure 6 for the new administrative capitalâs construction.5\n\nThe city will be established in three phases: Phase one is 40,000 acres, phase two is 47,000 acres, and phase three is 97,000 acres. Moreover, the NAC project went through modified phases during its construction as shown in Figure 7 and Table 8. These land use maps show that the residential and commercial and green areas are shrinking, which could be attributed to a financial investment, since real estate businesses frequently want higher building heights to obtain a piece of land, as well as acknowledgement of the infrastructureâs ability to support higher building heights. Thus, due to changes in density distribution, areas differ while revising phase construction maps, resulting in the scheme being divided into homogeneous zones to achieve integration, interdependence, and a population/place balance.16\n\nThe NAC planners took into consideration implementing a strong road and transport network in the proposed plan as shown in Figure 8 to support the future vision of the city by linking it regionally with Cairo, in addition to linking it internally to a public transport network that attracts and settles the population. This plan is also integrated with the regional plan for Greater Cairo.16\n\nAll Figures 6, 7 & 8 are acquired from The ACUD with a permission and were edited by the researcher.\n\nRegarding transportation, there is a public transport network that connects railway tracks which have metro lines with buses and microbuses, and all proposals are related to the planning idea of the proposed general strategic plan and emphasize the importance of linking the city with all available public means and in stages commensurate with the proposed priorities. The NAC is covered with variable means of transportations as shown in Figure 10 as metro lines, mini-buses, monorail lines and Light Rail Transit train lines (LRT) which is considered an upgraded model of the old tram as it works using electricity. They cover all the valleys in phase one and ensure citizens can use them easily saving time and with not much effort to access these means of transportation.5\n\nSmart applications embedded in the NAC:\n\nThe company developed the following guidelines as a focus for the smart city initiative:\n\n1- Improving services for citizens\n\n2- Energy sources and utilities\n\n3- Roads and transportation\n\nE-Health and E-Education â All medical and educational facilities must have the proper connectivity through the FTTx or IoT network to guarantee the e-Health and e-Education services deliverability according to their scope of work. All hospitals and clinics will be interconnected through a unified system to ensure the quality of health services offered to citizens across the whole city.\n\nSmart cards â Access control, digital identity, and payments for all services such as utilities, parking, and transit will all be managed with a single card.\n\nPublic Wi-Fi â With focus on infrastructure and connectivity, free Wi-Fi will be provided in public areas to help citizens in business and governmental districts stay connected.\n\nPortable cell tower â The new administrative capital and the telecommunication company implemented the first sample of a shared portable cell tower shown in Figure 9 for five different mobile network companies to cope with 4G and the 5th generation.16\n\nPhotovoltaic (PV) system â The system will be an on-grid PV system shown in Figure 10 connected to the low voltage side of the project buildingsâ distribution substations, and 50% of the roof top of each building will be reserved for PV system. The system will be equipped with smart metering system connected to the City Operation Center (COC). No batteries are required due to the high cost and inefficiencies of batteries.16\n\nSmart meters â Smart meters are smart utility network systems that enable the management and operation of vital facilities (electricity water gas) with cost and distribution efficiency.\n\nSmart waste management â System components will be managed by the COC through IoT platform to ensure operational efficiency and waste reduction across the city.\n\nElectric car charging stations â The electric car charging stations will be built and distributed across the city to encourage the use of electric cars with the focus on saving the environment and reducing pollution.\n\nIntelligent traffic and public transportation â Live updates on traffic to ensure smooth and easy trips for all citizens and to manage traffic lights, bus stations, gates, speed limits, gas stations, emergency situations and road directions based on real-time information.\n\nSmart street light column â designed to be sustainable street column repeated each 250 meters as shown in Figure 11 with exchangeable units utilized as cameras, public Wi-Fi, public addresses, digital ads, environmental sensors, cell phone network strengthening units, charging electric cars and smart lighting.16\n\nSmart parking â The smart parking system will make it easier for citizens to discover parking places and to pay for and reserve them.\n\nThe new administrative capital is implementing smart applications in several areas as shown in Table 9 in seeking to be a smart city, as the evaluation of smart cities should compromise âsmart applicationsâ as a measuring indicator which measures the smart citiesâ performance.\n\n\nDiscussion\n\nCities tend to relocate their capitals to reduce the burdens on the old capital. This is the situation in our case study, which suffers from overpopulation and several accumulative problems, including pollution caused by transportation problems, which cause health problems and so on.\n\nA smart capital city is proposed as a solution to a deteriorated, overcrowded capital city because it incorporates information and communication technologies (ICT) using smart applications to improve the quality of public services like energy, transportation, and utilities. These smart applications cover all dimensions of the smart city to reduce resource consumption, waste, overall costs, and improve the quality of life for its citizens through smart technology.\n\nIt was discovered through an analysis of global smart city experiences, as shown in Table 6, that new smart cities require smart apps, which differ from one smart city to the next depending on their demands, but all contribute to improving the quality of smart city factors through improving city services.\n\nAs previously stated in Table 7, the value of the dimension, which each city focused on developing its related smart applications, takes higher value than other dimensions. This proves that âsmart applicationsâ affect the value of evaluation of the âsmart city dimensionsâ, therefore affecting the whole city smartness evaluation. Thus, studying successful examples facing similar challenges gave a push towards developing Viennaâs evaluation criteria forming a suitable framework with suitable indicator which could be used in evaluating smart cities implementing smart applications as the NAC.\n\nTable 10 compares the different studied examples to the NAC according to the smart cityâs dimensions, their related factors, and the different smart applications fulfilling these factors, in addition to the percentages given by Vienna Regional Science Center standards (VRSCS) to evaluate the criteria of smart citiesâ performance mentioned earlier in the literature review.\n\nSmart people, smart governance, smart transportation, smart environment, and smart living are the six dimensions covered by the new administrative capital. It does not, however, specify any smart applications that address aspects of the smart economy. Smart cities should always strive for forward progress and integration of new smart technologies, in addition to existing ones, in order to keep up with the progress of smart cities. Although the number of indicators varies for each dimension in the Vienna framework, their relative weights were equal, which lacks neutrality; so relative weights should have different values.\n\n\nConclusions\n\nTo utilize Viennaâs framework to evaluate the NAC, the criteria require that the dimensions be given an acceptable weight, as neither the indicators, factors, or dimensions could have ever given equal values or had a similar impact on the cityâs performance.\n\nAs shown in Table 11, by applying a simple mathematical equation to the Standards for the Regional Science Center at the Technical University of Vienna, using the total average of the seven pre-evaluated cities and by taking into consideration that the total relative weight equals 100% as assumed in the criteria, the relative weight of each dimension gives the values in the last column. The final framework to evaluate smart cities after modification is shown in Table 12.\n\nResearchers could test the performance of implemented smart applications through workshops and questionnaires after the new capital operation, and then assign a score to each of the six smart city dimensions based on the established relative weight, allowing the smartness of the NAC to be evaluated.",
"appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nMendeley: Smart Capital City, https://data.mendeley.com/datasets/kcc2nwskb9. 17\n\nThis project contains the following extended data:\n\n⢠Figure 1 shows six core domains of a Smart City.\n\n⢠Figure 2 shows Structuring the analysis.\n\n⢠Figure 3 shows Characteristics and factors of smart city.\n\n⢠Figure 4 shows Application of the Vienna Technical University.\n\n⢠Figure 5 shows Final rating and composition.\n\n⢠Figure 6 shows NAC location.\n\n⢠Figure 7 shows 2016 Land use map.\n\n⢠Figure 7 shows 2021 Land use map.\n\n⢠Figure 8 shows NAC street network map.\n\n⢠Figure 8 shows Phase one means of transportation map.\n\n⢠Figure 9 shows Portable cell tower.\n\n⢠Figure 10 shows PVs on Ministries buildingsâ roofs.\n\n⢠Figure 11 shows Smart Street light column.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nReferences\n\nEl-Shakhs S: SADAT CITY, EGYPT AND THE ROLE OF NEW TOWN PLANNING IN THE DEVELOPING WORLD. Journal of Architectural and Planning Research. n.d.; Vol. 11(N, 21). Reference Source\n\nHahn J: Cities of tomorrow Challenges, visions, ways forward.2011. (Issue October). Publisher Full Text\n\nGiffinger R, Fertner C, Kramar H, et al.: City-ranking of European medium-sized cities. Centre of Regional Science: Vienna UT; 2007 October. Reference Source\n\nSadek EK: The Role Of Smart Cities In Solving Urban Problems (Case Study: Transportation Problems In Damascus). Damascus University. 2013; 2(29): 125â126. Reference Source\n\nEissa H, El-Nahas A: A Proposed Model for Measuring the Performance of Smart Cities in Egypt. ERJ. Engineering Research Journal. 2021; 44(1): 51â71. Publisher Full Text\n\nYusof N, van Loon J : Engineering a Global City: The Case of Cyberjaya. SAGE. n.d.; 15(4). Publisher Full Text\n\nLee SK, Kwon HR, Cho H, et al.: International Case Studies of Smart Cities: Singapore, Republic of Singapore. IDB-KRIHS Joint Research. 2016. June. Reference Source\n\nKim C: Place promotion and symbolic characterization of New Songdo City, South Korea. Elsevier; 2010; 27(1). Publisher Full Text\n\nDubai U, Selim G: The Role of the Information Technology on changing the Urban Form in2003. April 2003. Reference Source\n\nAsirvatham D, Brohi SN, Xion TE, et al.: Smart Mobility Cities: Connecting Bristol and Kuala Lumpur project report. 2018. October. Publisher Full Text\n\nBristol City Council: Connecting Bristol. 2019. Reference Source\n\nThe City Planning Administration: Stockholm City Plan. 2018; pp. 1â86. Reference Source\n\nHÀmÀlÀinen M: A Framework for a Smart City Design: Digital Transformation in the Helsinki Smart City. In: Ratten V, editor. Contributions to Management Science. Springer: Cham; 2020 January; pp. 63â86. Publisher Full Text\n\nThe Most Functional City in the World: Helsinki City Strategy 2017-2021. 2017. Reference Source\n\nYoussef H, Osman M, Roudi-Fahimi F: Responding to Rapid Population Growth in Egypt. Population Reference Bureau Policy Brief. 2014. Reference Source\n\nACUD: 2022. Reference Source\n\nIbrahim N: Smart Capital City. [Dataset]. Mendeley Data. 2023; V1. Publisher Full Text Reference Source"
}
|
[
{
"id": "170270",
"date": "03 May 2023",
"name": "Hebatullah Ghalib",
"expertise": [
"Reviewer Expertise Regional Urban Planning",
"Urban Design",
"Capital City",
"Development Strategies",
"Political Urbanism"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article title implies focus on the NAC as a case study for smart capital cities implementation. However, there is very little focus on the NAC. The article is more focused on smart cities in general and refers to two existing models of smart city assessment. The article also addresses several smart city examples to provide a comparative study. The authors use one assessment model for some cities and use the other model for the rest. There is no justification for this nor is there a criticism or comparison between the two models. The NAC is only addressed in one model which is later ignored in the conclusion. The conclusion focuses on the second model and states that its weighing of elements needs modification to be acceptable. There is no sufficient evidence on why they find the existing weight unacceptable or why their suggested weights are more acceptable. Finally, they suggest that the new weights can be used for future assessment of the NAC i.e. it hasn't yet been applied to the main case study of the paper.\n\nDetailed comments on each section of the article are as follows:\nAbstract\nI suggest revisiting and rephrasing most of the abstract as it can be misleading to the audience in terms of research objective and context of the case study.\nâStudying Egypt's overall urban development tendencies,âŠ. developing new urban communities to accommodate this increase is a requirement.â Actually, Egypt and especially Cairo has been developing new urban communities for decades now. Yet, they struggle to attract their target population and the informal settlements keep increasing. What you mention as a requirement is a continuous practice that has not solved the aforementioned challenges of migration, deteriorating healthcare and transportation. âThis paper discusses the strategy for transforming the old Cairo capital into a new administrative capital (NAC),â this statement is misleading as Cairo is not transforming into NAC. NAC is a new development in a desert location around 45 Km from Cairo. This means the objective of your paper is not clearly interpreted.\nIntroduction\nInternal migration from rural settlements to Cairo is a continuous phenomenon not just a sudden event in the 1950s.\nâsteady population increaseâ implies a constant rate of increase, is this the case here?\nWhy is lack of affordable housing seen as a follow up to loss of âenvironmental balanceâ?\nHow will the paper achieve its objective âensure that smart cities make services in the city more efficientâ?\n\nBackground\nKindly provide references for your historical background on smart city concept development and popularization.\nKindly provide references that support your statement on goals of smart communities.\nHow are âtransportation, economy, natural resources,âŠâ considered âtraditional urban development and growth theoriesâ? Kindly clarify or rephrase.\nâinnovation by cities (new, existing, or districts)â are you classifying cities as newly developed vs existing or looking into specific administrative levels e.g. districts? Kindly clarify.\nThe paper needs proofreading for better sentence phrasing to maintain a coherent line of thought and reorganize the sentences into balanced paragraphs instead of 2-sentence paragraphs.\nThe factors defining the smart city dimensions are mentioned in the text as 33 and in figure 2 as 31. Kindly verify and provide consistent information.\nThe figures show six dimensions, but they are mentioned in the text merely as a number rather than explained. Only three of them are very briefly explained in the text through a single sentence for each.\nGlobal classifications\nThis part needs proofreading to ensure clarity and use of suitable terminology.\nThe introduction of the discussed tools is inconsistent. It is preferable to be consistent with the aspects you discuss in each tool as it justifies your argument, discussion, and implementation.\nKindly provide more references to support your discussion of these tools.\nReferring to the MDGs is considered obsolete unless properly justified and related as the global commitment now is towards the SDGs.\nIn the background section you mentioned a total number of 74 indicators for smart city dimensions. In this part you state that there are â74 indicators to analyze the performance of each factorâ which implies 74 indicators per dimension. Kindly clarify. âcities with smart industries, industries that use technologies in production processesâ the term smart industries seems to include the latter industries. If thereâs a difference, kindly clarify.\nIn the background section you stated that smart people are not only defined by their level of education but here you state âcities that seek to develop the education system, in other words, the city with the most intelligent peopleâ. Kindly rephrase for better comprehension.\nâsmart city in order to reduce resource consumptionâŠcould be a solution given to a deteriorated overcrowded capital cityâ Kindly elaborate as this was introduced as the main concept of the paper in the abstract but is somewhat understated throughout the paper.\nâStudying successful examples that have faced comparable issuesâ Kindly explain the comparable issues on which you base your selection of examples as well as their analysis.\nRelated work\nThis subtitle should be changed to reflect the content and flow within the paper structure.\nKindly provide adequate references to support your statements.\nKindly elaborate on the choice of case studies and how they relate to the main issue you identified in your case study i.e. being an overcrowded capital city.\nDiscussion of the digital divide is very important and critical especially in countries of the global south with high levels of illiteracy. However, it is very briefly mentioned in one sentence in only one of your examples.\nWhy are the first three cities assessed using a generic qualitative list while the rest are assessed on a quantitative basis?\nAre tables 3,4 and 5 developed by the author or obtained from a reference? Kindly clarify.\nTable 7 is merely a compilation of tables 3,4,5. Table 7 sufficiently provides the information as tables 3,4,5 had only one row each so their compilation is more logical.\nThe figures for NAC precede the subtitle that indicates the beginning of the case of NAC. Kindly re-organize for a more coherent sequence.\nâarea south of Cairo Suez shown in..â I believe you mean south of Cairo-Suez Road. Kindly proofread to ensure accuracy and clarity.\nâoverpopulation crisis in Cairo and by the increasingly swelling number of residents and the continuous shrinking number of resourcesâ I believe overpopulation and âswellingâ number of residents are redundant. The use of the terms swelling and shrinking here is somewhat unusual and misleading. Kindly consider rephrasing.\nWhat are your criteria for labeling the road and transportation network as âstrongâ?\nThe reference you provided (16) doesnât include information on the NAC integration with the regional plan for greater Cairo that you mentioned.\nWhy is it 4G and 5th Generation and not 4G and 5G?\nDiscussion\nTable 10\nHow is âenvironment transportation systemâ a smart application for international accessibility?\nIn smart people, Songdo, Dubai and NAC seem to have e-learning covering all aspects. Your paper doesnât mention that e-learning covers aspects of lifelong learning and ethnic plurality especially in NAC. Kindly elaborate on this point.\nIn smart living, for education facilities, Songdo and Dubai have e-learning but NAC doesnât. This contradicts with the previous mention of e-learning in NAC. Kindly review for consistency or clarify the differences between them.\nWhy do equal weights in the Vienna framework show lack of neutrality? Why are your new proposed weights more neutral?\nWhy are you using the smart city dimensions for analysis in parallel with the Vienna framework yet keep them separate and apply them to different examples? There should be a justification for this and possibly a criticism/ analysis of the methodology of each.\nConclusion\nâTo utilize Viennaâs framework to evaluate the NAC, the criteria require that the dimensions be given an acceptable weight,..â How do you identify and justify this âacceptable weightâ? The application of the Vienna framework on NAC is not included in the paper nor is a conclusion on how the difference between the old weights and the âacceptable weightâ affect your analysis. These new weights can be better described as a recommendation for testing in future research.\nThe conclusion is focused on the change in weights in Vienna framework and disregards the other qualitative evaluation applied on the first three case studies which was actually applied on the NAC.\nHow does this reflect on your initial problem of Cairo suffering from overpopulation and resource shortages?\nThe conclusion needs to be more elaborate and to highlight the overall study results especially those related to the case mentioned in the paper title i.e. the NAC.\nFigures\nFigure 1 is not beneficial nor of added value as the information in it can be simply listed in the text.\nThe information in figure 3 can be simply and clearly provided in a table.\nFigure 4 is of low quality so itâs difficult to read. Also, the label mentions 7 cities but the figure seems to include more than 10 cities.\nFigure 6,7 are of low resolution, so they are illegible. Also, it isnât indicated whether they are the authorâs work or obtained from a reference. Same goes for figures 9,10,11.\nTwo maps are labeled as figure 7 which is confusing. Preferable to assign a separate figure label for each map for easier tracking of information. The same goes for figure 8.\nReferences\nThere are several peer-reviewed papers already published discussing the NAC that you can refer to for reliable data on the case study.\nThe argument and examination of case studies require more references for increased data reliability and ensured validity.\n\nIs the background of the caseâs history and progression described in sufficient detail? No\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Partly",
"responses": []
},
{
"id": "338978",
"date": "09 Dec 2024",
"name": "Oluwagbemiga Paul Agboola",
"expertise": [
"Reviewer Expertise SMART CITY DEVELOPMENT",
"URBAN RESILIENCE",
"CITY'S ADAPTATIONS",
"CITY'SÂ PLANNING",
"SMARTÂ TECHNOLOGY"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview for âSmart capital cities: towards a smart new administrative capital (NAC)â  This manuscript explores the challenges faced by Greater Cairo due to rapid urbanization, overpopulation, and resource shortages, particularly regarding migration, healthcare deterioration, and transportation issues. The paper emphasizes the need to develop new urban communities to accommodate the growing population and improve the quality of life. To improve the manuscript consider the following suggestions: 1. Clarify the Research Question/Problem Statement: Begin by refining the central research question or problem. This will make the purpose of the study clearer. For example: \"This paper explores the challenges posed by rapid urbanization in Greater Cairo and evaluates the strategy for transforming old Cairo into a new administrative capital (NAC) that aligns with the principles of a smart city.\" 2. Provide More Background Context: Expand on Greater Cairo's issues, such as overpopulation, migration, healthcare deterioration, and transportation problems. Provide more statistics or real-life examples to back up your claims and set the stage for your research. Mention previous studies or urban planning strategies in other cities for comparison to show how the proposed transformation of Cairo fits into global trends. 3. More reviews of the literature on  \"Smart City\": (i) Provide a more comprehensive definition of a \"smart city.\"(ii) A smart capital city is proposed as a solution to a deteriorated, overcrowded capital city because it incorporates information and communication technologies (ICT) using smart applications to improve the quality of public services like energy, transportation, and utilities. These smart applications cover all dimensions of the smart city to reduce resource consumption, waste, and overall costs, and improve the quality of life for its citizens through smart technology, (iii) It was discovered through an analysis of global smart city experiences, as shown in Table 6, that new smart cities require smart apps, which differ from one smart city to the next depending on their demands, but all contribute to improving the quality of smart city factors through improving city services, (ıv)  Include specific examples of smart technologies and their potential impact on urban development, (v)The goal of smart communities is to promote cities that provide basic infrastructure and provide a fair quality of life for their residents, as well as a clean and sustainable environment using âsmart applicationâ solutions. Items (i) to (v) extracted from the study should enhanced by reviewing and citing the following published manuscripts (i)Ref 1\n\n(ii) Ref 2\n\n(iii) Ref 3\n\n(iv) Ref 4 I strongly recommend these published manuscripts for references based on their relevance and contribution to the research under review, as they provide critical insights and updated findings.\n\n4. Highlight the Research Methodology: Mention briefly in the ABSTRACT how the research will evaluate the strategy for transforming Cairo into a smart city. Also, mention the âData collection processâ. 5. Emphasize the Benefits and Challenges of Implementation: Add a section that evaluates the potential benefits of the NAC transformation, such as improved quality of life, better services, and more sustainable urban planning. Then, balance this by acknowledging the challenges of implementing smart city technologies (e.g., funding, infrastructure, public resistance). 6. Improvement Ä°n Writing Style And Proofread The Entire Manuscript To Correct Some Grammatical Errors: Reword some sentences for clarity and flow. For example, âCities are rapidly evolving, expanding in size and density while confronting major challenges such as migration, deteriorating healthcare, and transportation issues.â could be more concise: \"Cities are rapidly expanding in both size and density, facing challenges like migration, deteriorating healthcare, and transportation congestion.\" Use more varied sentence structures to keep the reader engaged. 7. Incorporate a Conclusion and Future Directions: â¢\n\nAdd a conclusion section to summarize the findings or discussions from the paper. It should tie back to the research question and highlight the key takeaways. â¢\n\nDiscuss potential future research directions or recommendations for the city of Cairo.\n\nBy refining these aspects of your manuscript, you'll enhance the clarity, depth, and relevance of the research, ensuring it resonates with readers interested in urban planning and smart city transformations\n\nIs the background of the caseâs history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes",
"responses": []
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https://f1000research.com/articles/12-265
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https://f1000research.com/articles/12-264/v1
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13 Mar 23
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{
"type": "Research Article",
"title": "Evaluation of the dimensional changes in the mandible, condyles, and the temporomandibular joint following skeletal class III treatment with chin cup and bonded maxillary bite block using low-dose computed tomography: A single-center, randomized controlled trial",
"authors": [
"Amr H. Husson",
"Ahmad S. Burhan",
"Mohammad Younis Hajeer",
"Fehmieh R. Nawaya",
"Amr H. Husson",
"Ahmad S. Burhan",
"Fehmieh R. Nawaya"
],
"abstract": "Background: Insufficient evidence regarding the effects of chincup therapy on the mandibular dimensions and temporomandibular joint (TMJ) structures requires high-quality studies using three-dimensional (3D) imaging. This trial aimed to evaluate the 3D changes in the mandible, condyles, and glenoid fossa after chin cup therapy for skeletal Class III children compared to untreated controls. Methods: A 2-arm parallel-group randomized controlled trial on 38 prognathic children (21 boys and 17 girls), with mean ages 6.63±0.84 years. Patients were recruited and randomized into two equal groups; the experimental group (CC) was treated with occipital-traction chin cups in conjunction with bonded maxillary bite blocks. No treatment was provided in the control group (CON). Low-dose CT images were acquired before (T1) and after achieving (2-4 mm) positive overjet (T2), and after 16 months apart in both groups. The outcome measures of the condyle-mandibular 3D distances, the condyles-glenoid fossa postional changes, and the quantitative displacement parameters of superimposed 3D models were compared statistically. Paired- and two-sample t-tests were used for intra- and inter-group comparisons, respectively. Results: Overall, 35 patients (18 and 17 in the CC and the CON groups, respetively) were enrolled in the statistical analysis. The mean mandibular and condylar volumes increased significantly by 777.24 mm3 and 1,221.62 mm3, 94.57 mm3, and 132.54 mm3 in the CC and CON groups, respectively. No statistically significant differences were observed between the groups regarding the volumes, superficial areas, and linear changes of the mandible and condyles, and part analysis measurements, except the changes of the relative sagittal and vertical positions of condyles, glenoid fossa, and posterior joint space, which were significantly smaller in the CC group (p<0.05) than the CON group. Conclusions: The chin cup did not affect the mandibular dimensions. Its primary action was confined to the condyles and the TMJ internal dimensions. Clinicaltrials.gov registration: NCT05350306 (28/04/2022).",
"keywords": [
"Skeletal Class III malocclusion",
"chin cup",
"low-dose computed tomography",
"lose-dose CT",
"volumetric assessment"
],
"content": "Introduction\n\nThe efficacy of the chin cup in controlling mandibular growth remains a controversial matter.1,2 Findings in some clinical studies support retardation or restriction of mandibular growth,3â5 but others show the opposite.6,7 Still, other studies highlight only changes in shape and redirection of mandibular growth.8,9 The conclusion of Liu's2 systematic review indicated insufficient data to judge the efficacy of the chin cup on mandibular inhibition. A recent systematic review by Chatzoudi1 concluded that more high-quality studies with proper methodology, untreated control groups, and reliable measurements are needed. Moreover, the supposed changes in the temporomandibular joint (TMJ) due to the retraction force applied by a chin cup is not yet clear. Zurfluh10 stated that the low-quality data from past studies hindered pointing out clear statements regarding the effects of chin-cup treatment on TMJ. Nevertheless, TMJ changes associated with this treatment need to be evaluated.\n\nPreviously, mandibular adaptation to chin cup force has been evaluated using two-dimensional (2D) lateral cephalograms or panoramic radiographs. However, the information provided by these techniques is limited in evaluating the condyles and mandibular ramus.11 Therefore, the three-dimensional (3D) analysis using 3D computed tomography (CT) or cone-beam CT (CBCT) has become important.12 Although CBCT uses relatively low radiation compared to conventional CT, the accuracy of this technique is still doubtful in detecting condylar changes.13,14 Low-dose CT is an alternative reliable procedure with an effective dose approximately equal to traditional radiographs.15 This technique has been used in some clinical studies.16,17\n\nHistologically, experimental animal studies have demonstrated a reduction in the width of the prechondroblastic zone and a decrease in the cellularity of the condylar head after chin cup therapy.18 In this respect, investigating the volumetric changes in condyles may be useful.\n\nTo our knowledge, no study has investigated the volumetric changes of the mandible and condyles after early chin cup therapy. This randomized controlled trial aimed to evaluate the changes in the mandible, condyles, and glenoid fossa following the chincup therapy of Class III malocclusion patients in comparison with those changes in a control group of untreated patients. It aimed to test the null hypotheses that no significant differences existed between the chincup and the control group concerning the changes in the mandible, the condyles, and the glenoid fossa.\n\n\nMethods\n\nThe research was designed as a single-center, 2-arm, parallel-group randomized controlled trial with a 1:1 allocation ratio and guided by the CONSORT statement.19 The study was conducted in the Department of Orthodontics at the University of Damascus Dental School between January 2019 to January 2022. This study was approved by the Regional Ethical Committee on Research of the Damascus University, Faculty of Dentistry, Syria (UDDS-2788-2016PG) on January 11, 2019. The study was registered at ClinicalTrails.gov (Identifier: NCT05350306) on 28 April, 2022 after the onset of the study. Retrospective registration was done as the regulations at the Univeristy of Damascus do not oblige researchers to register their trial protocols before the commencement of their research work. However, this protocol was registered retrospectively to publish the trial's results. This article is reported in line with the CONSORT guidelines.44\n\nSample estimation was calculated using Minitab® Version 18 (Minitab Inc., State College, Pennsylvania, USA). An independent t-test, statistical power of 85%, and a significance level of 0.05 were among the assumptions for this calculation. In total, 17 children were required in each group to detect a 1 mm significant difference in total mandibular length (Co-Gn) based on the standard deviation of this measurement for a previous study (0.92).20 Four patients were added to the experimental and control groups (2 patients in each group) to compensate for potential dropouts.\n\nA preliminary screening was performed of schoolchildren during the period from April 2019 to May 2019. Ten public and private schools were randomly selected from the database of all primary schools in Damascus city, Syria. Intra- and extra-oral clinical examination was performed by the principal investigator (A.H.H.) for 1700 children with an average age of 7 years (range: 6-8 years), including 767 female and 933 male children at the medical clinics of the selected schools. Initially, 90 children with class III malocclusion (52 boys, 43 girls) were expected to be suitable for inclusion in the trial. A formal invitation letter was sent to the parents of candidate children asking them to visit the Department of Orthodontics at the University of Damascus for further analysis and to assess the need for early correction of the deformity. All invited parents came for this in-depth assessment. The following inclusion criteria were used: age between 6 and 8, edge-to-edge incisor relationship or anterior crossbite, class III relationships at the permanent first molars or mesial-step relationships at primary second molars, absence of discrepancy between the centric relation and the maximum intercuspation position, short or normal-face pattern, normal or deep overbite, and no temporomandibular joint disorders or craniofacial anomalies. The radiological inclusion criteria were (1) mild to moderate mandibular prognathism (SNB > 80°, and 0 â€ANBâ¥4) and (2) normal or horizontal growth pattern (Bjorkâs sum â€396° ±5°). Fifty seven patients met the inclusion criteria. Information sheets were given to parents of eligible patients. The need for two low-dose CT images, and the possibility of postponing treatment if the child fell into the control group were elaborately explained before taking their written informed consents. Six parents refused to participate in this trial. Therefore the resultant sampling frame included 51 patients. Simple random sampling was used to select 38 patients for this trial who were then assigned randomly to the chin cup and the control groups. The CONSORT flow diagram of patientsâ recruitment, follow-up, and entry into data analysis is given in Figure 1.\n\nAn online randomization service (www.randomizer.org) was used to perform simple randomization with an allocation ratio of 1:1. The allocation sequence was concealed from the researcher (AH) using sequentially numbered, opaque, and sealed envelopes, which were opened after patients completed all baseline assessments. Blinding of either patient or operator was not possible. Blinding was applied for the outcome assessment only. After the mandibular mask was constructed by one of the co-authors (MYH), the row image was resliced by cutting the region of the overjet to blind the other investigator (AH), who completed the extraction of results.\n\nInterventions\n\nExperimental group: Chin-cup group\n\nEach patient in the treatment group received a bonded maxillary bite block and an occipital chin cup (Figure 2 shows the bite block). The splint consisted of a 2 mm posterior acrylic cap reinforced by a metal framework. The traction force on the mandible by the chin cup was 400â500 g at each side, in the direction of the condyles.8 Patients were instructed to wear the appliance for 14 hours per day. The true overjet and overbite were measured monthly, subtraction the effects of the bite block on these variables based on the assumption that every 1 mm of the thickness of the posterior bite block increases the overjet by an average of 1.3 mm and decreases the overbite an average of 2 mm.21 After achieving 1 mm overjet and 2 mm overbite, the acrylic splint was removed. Then, the chin cup was used with a light force for approximately 8 hours per day as a retainer. The active treatment was considered complete when the following two conditions were met: a positive overjet of 2 to 4 mm, and an active treatment time of at least 16 months.\n\nAfter acquiring low-dose CT images, the patients allocated to the control group received no clinical intervention. They were recalled 16 months after registration for collection of the second low-dose CT image records.\n\nComputed tomography acquisition\n\nA multiplanar spiral CT machine (Philips brilliance 64, Philips Medical Systems, Best, the Netherlands) was used to obtain pre- (T1) and post-treatment (T2) CT images. The CT scans were performed at 80 kV and 100 mAs, one pitch, 2.5 mGy (CTDIvol), and a 1.25 mm slice thickness.17 The patients were seated in a supine position with their Frankfort horizontal (FH) plane perpendicular to the floor and their teeth in maximum intercuspation. Once the low-dose CT images were stored in the Digital Imaging and Communications in Medicine (DICOM) format, they were transferred into MIMICS 21.0 software (Materialise, Leuven, Belgium) to create 3D volumetric mandibular models.\n\nMandible and condyles reconstruction\n\nEach image was resliced to make the head orientation uniform along the midsagittal and Frankfort horizontal planes. The basion, crista galli, and glabella landmarks defined the midsagittal plane. The Frankfort horizontal plane was defined by the right and left orbitale landmarks and the right porion landmark.22\n\nThe image processing procedures are shown in Figure 3. Briefly, a bony mask was created using a global threshold value of 226 to 2976 Hounsfield units [HU] for the software algorithm. Semiautomatic segmentation using a split mask tool was utilized to isolate the mandibular bone. The mandible mask was rendered in a high-quality 3D model. Due to the differences in the lower permanent molar eruption, the crowns of the teeth were removed by a plane passing through 1 mm inferior to the alveolar bone and 10 mm distal to the second primary molars. Finally, the condyles were cut at neck level using a plane parallel to the Frankfort horizontal plane at the most inferior part of the sigmoid notch, and the post-treatment cut was parallel to the first cut.23 The condyle and mandible volumes and surface sizes were automatically calculated in mm3 and mm2, respectively.\n\nLinear and angular measurements\n\nA new template was created from the Measure and Analyze tool of Mimicsâ¢, which was used to define the anatomical landmarks and planes and produce the desired measurements (Tables 1 and 2).22,24,25 Ikeda and Kawamura suggested that the condylar and glenoid fossa landmarks were made on the corrected sagittal view.26 After identifying the landmarks, the software automatically calculated the distances and the angles. Then, the data was exported in.cvs format.\n\n* Definitions of these measurement are taken from: Mavreas and Athanasiou,24 Alhammadi et al.,22 and Celikoglu et al.25\n\n* Modified from: Mavreas and Athanasiou,24 Alhammadi et al.,22 and Celikoglu et al.25\n\n3D mandibular regional superimposition and comparison analysis\n\nThe paired resulting reconstructed mandibular models were exported to 3-Matic software (3-matic13.0, Materialise NV, Leuven, Belgium), and their surfaces were warped using the Warp tool of Mimicsâ¢. Initially, The T1 and T2 3D volumes were superimposed manually by approximating similar anatomical regions of the mandible using Interactive Translate and Rotate tools of the software, followed by automatic global registration in 3-Matic software (Figure 4). The superimposition was repeated three times to enhance accuracy and reproducibility. The point-based analysis was performed to assess the changes in 3D mandibular models between T1 and T2, and a color map was produced to assess the mandibular shape changes. The threshold was set at 2 mm: green areas indicated differences within 2 mm (between â2 and 2 mm), red surfaces indicated positive values displacement more than 2 mm, and blue surfaces indicated negative values displacement greater than â2 mm between two 3D models (Figure 5). Quantitative changes were done by reporting the mean, minimum, and maximum values of part analyses on a spreadsheet and used for comparative analyses\n\nInterim analyses and stopping guidelines\n\nNo interim analyses were performed during this trial.\n\nStatistical analyses were performed using IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., NY, USA). The average value of each bilateral measurement was calculated to achieve the statistics of this study. ShapiroâWilk test was used to test the normality of data. Accordingly, non-normally distributed data were analyzed by the Wilcoxon signed-rank test and the MannâWhitney U test for in- and intergroup comparison, respectively. Additionally, a paired-samples t-test was used to evaluate the changes in the treatment group, and an independent t-test was used for the intergroup comparisons of parameters with a normal distribution. Bonferroni's correction was used to adjust the alpha level due to multiplicity. The adjusted alpha level was 0.0125 for the volumetric and surface area measurements and 0.005 for the cephalometric analysis. To assess the reliability of the measurements, 17 (25%) images were randomly selected and re-measured after a 2-week interval by the same examiner (AH). Intraclass correlation coefficients (ICCs) with the absolute agreement were used to assess intra-rater reliability. Error of the method was analyzed with Dahlberg's formula.27\n\n\nResults\n\nIntra-examiner reliability was high, and ICCs ranged from 0.83 to 0.999. The error of the method was acceptable for both linear and angular measurements, which were smaller than 0.5 mm and 0.6 degrees, respectively. The measurement error ranged from 18.84 to 54.3 mm3 for the volumetric measurements and 18.81 to 44.58 mm2 for the surface area measurements (Table 3).\n\n* Mean differences between the two measures.\n\n** Evaluated using the Dahlberg's formula.27\n\nBasic sample characteristics\n\nThe CONSORT flow diagram of patients' recruitment and follow-up is given in Figure 1. In total, 38 patients (21 boys and 17 girls) were enrolled.44 However, three patients were not included in the statistical analysis: one in each group was lost to follow-up, and the third one was extracted from the control group due to the low-quality low-dose CT image. Table 4 shows the basic sample characteristics. The mean treatment and observation periods were 16.67±0.52 months and 16.67±0.52 months, respectively. There were no significant differences in the age distribution and treatment/observation periods between the experimental and the control groups (p=0.675 and p=0.179, respectively).\n\nâ Pearson's Chi-square test.\n\nâ¡ Two-sample t-test.\n\nThe mean mandibular volume increased significantly by 777.24 mm3 and 1,221.62 mm3 in the chin cup and control groups, respectively. Also, the mean condylar volume increased significantly by 94.57 mm3 and 132.54 mm3 in the chin cup and control groups, respectively (Table 5). The linear measurements of the mandible significantly increased in both groups (p<0.05). No statistically significant differences between the chin cup and control groups were observed regarding the volumes, superficial areas, and linear changes of the mandible and condyles (Tables 5 and 6). Although the mean mandibular angle decreased by 1.63° in the treatment group, there was no statistically significant difference between the treatment and the control groups regarding this variable (Table 6).\n\nâ Paired t-test.\n\nâ¡ Wilcoxon signed-rank test.\n\n§ Two-sample t test.\n\nâ Mann-Whitney U test.\n\n* Statistically significant at p<0.0125.\n\nâ Paired t-test.\n\nâ¡ Wilcoxon signed-rank test.\n\n§ Two-sample t test.\n\nâ Mann-Whitney U test.\n\n* Statistically significant at p<0.005.\n\nThere was a significant posterior and superior displacement of the condyles following treatment by a mean of 1.29 mm (p=0.001) and 1.68 mm (p<0.001), respectively. Similar changes were observed in the glenoid fossa, but to a lesser extent by a mean of 0.82 mm posteriorly (p<0.001) and 0.81mm superiorly (p<0.001). The superior joint space (SJS) and posterior joint space (PJS) showed a significant mean decrease of 0.71 mm and 0.93 mm, respectively. However, no statistically significant differences were observed in the relative positions of the condyles, glenoid fossa, and joint spaces in the control group (p>0.05), except for a significantly superior displacement of the condyles (0.84 mm; p=0.001). The changes in the relative sagittal and vertical positions of condyles and glenoid fossa were significantly smaller in the treatment group than in the control group. No statistically significant differences were observed between the treatment and control groups regarding the average mean, maximum, and minimum part analysis of the point-based analysis (p>0.05; Table 7).\n\nâ Mann-Whitney U test.\n\nâ¡ Two-sample tâtest.\n\nHarms\n\nNo serious harm was observed.\n\n\nDiscussion\n\nAlthough the extensive literature on the potential condyle-mandibular adaptation of the chin cup is of interest, the conflicting outcomes urge high-quality studies that apply 3D imaging technology to the study of the TMJ, such as CBCT or CT scan.1,2,12 But, the difficulty achieving condylar segmentation in CBCT images is due to its lesser density compared to the rest of the mandible, as demonstrated previously.28 Therefore, the use of low-dose CT imaging was preferred in this study. In addition, the acceptable radiation dose of this imaging method is 0.51 mSv under the acceptable limit of 1 mSv per year.13,15\n\nPrecise volumetric assessment can be affected by several variables, such as voxel size, patient position, and the characteristics of the software used for segmentation.28,29 The used software has been proven by Weissheimer30 as the most accurate software with errors in the volume segmentations less than 2% of the real size.\n\nThe influence of sex on the treatment outcomes was not accounted for in this study. Baccetti31 found no sex differences in most dentofacial parameters for Class III patients until the age of 13 years. The mean age of the treatment group was 6.59 (±0.86) years, which has been considered early optimal treatment timing.32 Additionally, the mean age of the control group sample was 6.57 (±0.83) years, which gives the patients the time to be treated ideally after the observation period. The treatment period was 17.02 (±0.92) months. Mitani33 stated that the major treatment effects of the chin cup are observed in the first 2 to 3 years of treatment. Some previous studies suggested variable treatment periods from 1-7 years, but most stopped heavy forces after obtaining a good overjet. On the other hand, treatment effects can be better demonstrated if longer periods of observation were implemented in the current study.\n\nAlthough the force applied from the chin cup, the volume and the superficial area of the mandible showed a significant increase. These changes could be secondary to simultaneous bone repositioning on most surfaces of the mandible.34 Similarly, the linear mandibular measurements increased significantly in both groups. It was observed that the mean of the increase in the mandibular dimensions was smaller in the treatment group than in the untreated group, but no differences were found between both groups regarding these parameters. Due to several studies on chin cup therapy using 2D measurements based on cephalometric radiographs, only linear measurements of this trial could be compared. The results of this study regarding the changes in the linear mandibular measurements were in agreement with some relevant clinical studies.6â8 On the contrary, Mimura and Deguchi35 reported a similar increase in the mandibular length in the chin cup and control groups; these data suggest that chin cup use does not decrease the overall mandibular growth. As mentioned previously, the differences in the appliance design, the force application, and the evaluation method might cause differences in the results.\n\nThe gonial angle showed a closing pattern at the end of treatment. This gonial angle change has also been reported in the literature.7,8,36 Lin et al.37 attributed the reason to the force's direction that tends the mandible's ramus to rotate around the gonial angle.34 Nevertheless, the observed change in the gonial angle in this trial was insignificant in the chin cup group and among the groups. It is more likely to be affected by the individual growth pattern, as the gonial area has some freedom to remodel.8\n\nThe condyle's volume and superficial area increased significantly in both groups. It is important to highlight that even though no statistically significant differences were found, the overall changes in the condyles in the treatment group were smaller than in the control group. This change had been previously demonstrated by experimental studies showing a decrease in the condylar growth rate due to the compression force applied by the chin cup.18\n\nAnother point that should be considered is the observed extensive standard deviation in the volumetric condylar change. The individual differences in the condyles' size, form, and growth cartilage; thus, growth magnitude should also be individually different. In turn, the chin cup force affects condyles variably in the clinical environment.33\n\nThe distinguishing differences between the two groups involved most of the TMJ parts. The condyle was displaced posteriorly and superiorly in the chin cup group and accompanied by a posterior and posterior remodeling of the glenoid fossa. However, it showed lesser superior and posterior displacement than the condyle. The histological differences between the glenoid fossa and the condyles could be the reason for the non-equivalent changes.38 Interestingly, Pancherz et al.39 observed a later remodeling response of the glenoid fossa than that occurring in condyles after the Herbst appliance. The study findings regarding the condyles and glenoid fossa portions were similar to those of Mimura and Deguchi.35 However, neither the amounts of displacement nor the evaluation method was similar. On the contrary, Gökalp and Kurt40 showed no changes in condylar positioning by magnetic resonance imaging after chin cup therapy. The evaluation method, however, might cause differences in the results.\n\nIn this context, Lee et al.41 found that the facemask appliance induced similar effects but with a smaller magnitude. Results from Grandori42 could explain the difference, revealing that only 70% of the facemask force was translated to the chin. Surprisingly, De Clerk et al.43 used bone-anchored Class III intermaxillary traction with a force of 250 g/side, but the amounts of displacement of the condyle and glenoid fossa were greater than those observed in this study. These differences might be due to the duration of the applied force and the age of the sample in his study, which was older and closer to puberty than in this study.\n\nThe results indicated a decrease in the spaces of the joint, except for the anterior space. This may be attributed to the backward and upward forces applied from the chin cup, with the slow remodeling of the glenoid fossa, as mentioned previously.39 Gökalp and Kurtâs40 study showed no change in the condyle-glenoid fossa relationship. However, the evaluation method and the relatively small sample size make the comparison meaningless. These changes require long-term evaluation to corroborate the TMJ adaptation to these positional changes.\n\nThe analysis of the changes in mandibular shape through 3D superimposition showed no differences between the two groups, except some inward changes were noticed in the symphysis region, which is indicated by the blue areas in the color mapping in Figure 6. This type of change has been reported previously and might be induced by the plastic cup of chin cup.8,33 The condyles showed atypical changes. On the contrary, the past 2D cephalometric studies assumed that chin cup treatment could induce a forward bending of the condylar neck.8,35 The individual differences in growth direction and magnitude, and sometimes the asymmetric position of the condyles, could affect condylar reaction either to the chin cup or the growth.33\n\nQuantitative results of the point-based analysis showed positive mean displacement in both groups. The average mean and maximum point part analysis was smaller in the chin cup group than in the control group. Although intergroup comparisons according to the parameters of the point part analysis showed insignificant differences between the two groups, class III malocclusion worsens in untreated subjects over time; thus, early treatment is required.\n\nOne of the limitations of the current trial is its short-term evaluation. Secondly, the temporomandibular disk was not studied in this trial. However, this aim needs another imaging method.\n\nRandomized controlled trials (RCTs) provide the highest level of evidence.10 Additionally, three-dimensional quantification gives a true mandibular, condyle, and glenoid fossa changes after orthodontic treatment by the chin cup.43 Moreover, significant changes occur in the craniofacial region during the mixed dentition that can be utilized for orthodontic therapy.32 Our study designed as RCT with the help of the three-dimensional quantification and patients were in the early mixed dentation, as a result, the current research findings revealing the effects of the chincup are generalizable. In this randomized controlled trial, the chin cup treatment failed to control mandibular growth, resulting only in orientation to condylar positions and compression of joint spaces.\n\n\nConclusions\n\nIn conclusion, the chin cup seemed to have no effects on mandibular dimensions in this study. The major action of the retroactive force of the chin cup oriented onto the condylar postions and was associated with compression of the joint spaces.",
"appendix": "Data availablity\n\nFigshare: Evaluation of the Dimensional Changes in the Mandible, Condyles, and the Temporomandibular Joint Following Skeletal Class III Treatment with Chin Cup and Bonded Maxillary Bite Block Using Low-Dose Computed Tomography: A Single-center, Randomized Controlled Trial. https://doi.org/10.6084/m9.figshare.21973517.v3. 44\n\nThis project contains the following underlying data:\n\n- Data file 1: linear anlaysis.xlsx\n\n- Data file 2: part analysis.xlsx\n\n- Date file 3: volume and area analysis.xlsx\n\nFigshare: Evaluation of the Dimensional Changes in the Mandible, Condyles, and the Temporomandibular Joint Following Skeletal Class III Treatment with Chin Cup and Bonded Maxillary Bite Block Using Low-Dose Computed Tomography: A Single-center, Randomized Controlled Trial. https://doi.org/10.6084/m9.figshare.21973517.v3. 44\n\nThis project contains the following extended data:\n\n- Extended data file 1: Information sheet (in Arabic and English)\n\n- Extended data fille 2: Consent form (in Arabic and English)\n\nFigshare: CONSORT checklist for âEvaluation of the Dimensional Changes in the Mandible, Condyles, and the Temporomandibular Joint Following Skeletal Class III Treatment with Chin Cup and Bonded Maxillary Bite Block Using Low-Dose Computed Tomography: A Single-center, Randomized Controlled Trialâ. https://doi.org/10.6084/m9.figshare.21973517.v3. 44\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nChatzoudi MI, Ioannidou-Marathiotou I, Papadopoulos MA: Clinical effectiveness of chin cup treatment for the management of Class III malocclusion in pre-pubertal patients: a systematic review and meta-analysis. Prog. Orthod. 2014; 15(1): 62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu Z, Li C, Hu H, et al.: Efficacy of short-term chincup therapy for mandibular growth retardation in Class III malocclusionA systematic review. Angle Orthod. 2011; 81(1): 162â168. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeguchi T, Kuroda T, Minoshima Y, et al.: Craniofacial features of patients with Class III abnormalities: growth-related changes and effects of short-term and long-term chincup therapy. Am. J. Orthod. Dentofac. Orthop. 2002; 121(1): 84â92. PubMed Abstract | Publisher Full Text\n\nChang HP, Lin HC, Liu PH, et al.: Geometric morphometric assessment of treatment effects of maxillary protraction combined with chin cup appliance on the maxillofacial complex. J. Oral Rehabil. 2005; 32(10): 720â728. PubMed Abstract | Publisher Full Text\n\nGraber LW: Chin cup therapy for mandibular prognathism. Am. J. Orthod. 1977; 72(1): 23â41. Publisher Full Text\n\nTuncer BB, Kaygisiz E, Tuncer C, et al.: Pharyngeal airway dimensions after chin cup treatment in Class III malocclusion subjects. J. Oral Rehabil. 2009; 36(2): 110â117. PubMed Abstract | Publisher Full Text\n\nBarrett AAF, Baccetti T, Mcnamara JA, et al.: Treatment effects of the light-force chincup. Am. J. Orthod. Dentofac. Orthop. 2010; 138(4): 468â476. PubMed Abstract | Publisher Full Text\n\nDeguchi T, Mcnamara JA: Craniofacial adaptations induced by chincup therapy in Class III patients. Am. J. Orthod. Dentofac. Orthop. 1999; 115(2): 175â182. PubMed Abstract | Publisher Full Text\n\nAlarcon JA, Bastir M, Rosas A, et al.: Chincup treatment modifies the mandibular shape in children with prognathism. Am. J. Orthod. Dentofac. Orthop. 2011; 140(1): 38â43. PubMed Abstract | Publisher Full Text\n\nZurfluh MA, Kloukos D, Patcas R, et al.: Effect of chin-cup treatment on the temporomandibular joint: a systematic review. Eur. J. Orthod. 2015; 37(3): 314â324. PubMed Abstract | Publisher Full Text\n\nMarkic G, Muller L, Patcas R, et al.: Assessing the length of the mandibular ramus and the condylar process: a comparison of OPG, CBCT, CT, MRI, and lateral cephalometric measurements. Eur. J. Orthod. 2015; 37(1): 13â21. PubMed Abstract | Publisher Full Text\n\nSwennen GR, Schutyser F: Three-dimensional cephalometry: spiral multi-slice vs cone-beam computed tomography. Am. J. Orthod. Dentofac. Orthop. 2006; 130(3): 410â416. PubMed Abstract | Publisher Full Text\n\nHintze H, Wiese M, Wenzel A: Cone beam CT and conventional tomography for the detection of morphological temporomandibular joint changes. Dentomaxillofac. Radiol. 2007; 36(4): 192â197. PubMed Abstract | Publisher Full Text\n\nPatel A, Tee BC, Fields H, et al.: Evaluation of cone-beam computed tomography in the diagnosis of simulated small osseous defects in the mandibular condyle. Am. J. Orthod. Dentofac. Orthop. 2014; 145(2): 143â156. PubMed Abstract | Publisher Full Text\n\nCordasco G, Portelli M, Militi A, et al.: Low-dose protocol of the spiral CT in orthodontics: comparative evaluation of entrance skin dose with traditional X-ray techniques. Prog. Orthod. 2013; 14(1): 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCordasco G, Nucera R, Fastuca R, et al.: Effects of orthopedic maxillary expansion on nasal cavity size in growing subjects: a low dose computer tomography clinical trial. Int. J. Pediatr. Otorhinolaryngol. 2012; 76(11): 1547â1551. PubMed Abstract | Publisher Full Text\n\nBallanti F, Lione R, Fanucci E, et al.: Immediate and post-retention effects of rapid maxillary expansion investigated by computed tomography in growing patients. Angle Orthod. 2009; 79(1): 24â29. PubMed Abstract | Publisher Full Text\n\nPetrovic AG, Stutzmann JJ, Oudet CL: Control process in the postnatal growth of the condylar cartilage of the mandible. J. A MN, editor. Monography 4 Craniofacial Growth Series. Ann Arbor: Center for Human Growth and Development, University of Michigan; 1975; pp. 101â153.\n\nCampbell MK, Piaggio G, Elbourne DR, et al.: Consort 2010 statement: extension to cluster randomised trials. BMJ (Clinical research ed.). 2012; 345: e5661. Publisher Full Text\n\nUcuncu N, Ucem TT, Yuksel S: A comparison of chincap and maxillary protraction appliances in the treatment of skeletal Class III malocclusions. Eur. J. Orthod. 2000; 22(1): 43â51. PubMed Abstract | Publisher Full Text\n\nRebibo M, Darmouni L, Jouvin J, et al.: Vertical dimension of occlusion: the keys to decision. Int. J. Stomatol. Occlusion Med. 2009; 2(3): 147â159. Publisher Full Text\n\nAlhammadi MS, Shafey AS, Fayed MS, et al.: Temporomandibular joint measurements in normal occlusion: A three-dimensional cone beam computed tomography analysis. J. World Fed. Orthod. 2014; 3(4): 155â162. Publisher Full Text\n\nJiang YY, Sun L, Wang H, et al.: Three-dimensional cone beam computed tomography analysis of temporomandibular joint response to the Twin-block functional appliance. Korean J. Orthod. 2020; 50(2): 86â97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMavreas D, Athanasiou AE: Tomographic assessment of alterations of the temporomandibular joint after orthognathic surgery. Eur. J. Orthod. 1992; 14(1): 3â15. PubMed Abstract | Publisher Full Text\n\nCelikoglu M, Ucar FI, Buyuk SK, et al.: Evaluation of the mandibular volume and correlating variables in patients affected by unilateral and bilateral cleft lip and palate: a cone-beam computed tomography study. Clin. Oral Investig. 2016; 20(7): 1741â1746. PubMed Abstract | Publisher Full Text\n\nIkeda K, Kawamura A: Assessment of optimal condylar position with limited cone-beam computed tomography. Am. J. Orthod. Dentofac. Orthop. 2009; 135(4): 495â501. PubMed Abstract | Publisher Full Text\n\nDahlberg G: Statistical methods for medical and biological students. London: George Allen and Unwin; 1940.\n\nEngelbrecht WP, Fourie Z, Damstra J, et al.: The influence of the segmentation process on 3D measurements from cone beam computed tomography-derived surface models. Clin. Oral Investig. 2013; 17(8): 1919â1927. Publisher Full Text\n\nLo Giudice A, Ronsivalle V, Grippaudo C, et al.: One Step before 3D PrintingâEvaluation of Imaging Software Accuracy for 3-Dimensional Analysis of the Mandible: A Comparative Study Using a Surface-to-Surface Matching Technique. Materials (Basel). 2020; 13(12): 2798. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeissheimer A, Menezes LM, Sameshima GT, et al.: Imaging software accuracy for 3-dimensional analysis of the upper airway. Am. J. Orthod. Dentofac. Orthop. 2012; 142(6): 801â813. PubMed Abstract | Publisher Full Text\n\nBaccetti T, Reyes BC, Mcnamara JA, et al.: Gender differences in Class III malocclusion. Angle Orthod. 2005; 75(4): 510â520.\n\nSugawara J, Asano T, Endo N, et al.: Long-term effects of chincap therapy on skeletal profile in mandibular prognathism. Am. J. Orthod. Dentofac. Orthop. 1990; 98(2): 127â133. PubMed Abstract | Publisher Full Text\n\nMitani H: Recovery growth of the mandible after chin cup therapy: fact or fiction. Paper presented at: Semin Orthod. 2007.\n\nEnlow DH, Harris DB: A study of the postnatal growth of the human mandible. Am. J. Orthod. 1964; 50(1): 25â50. Publisher Full Text\n\nMimura H, Deguchi T: Morphologic adaptation of temporomandibular joint after chincup therapy. Am. J. Orthod. Dentofac. Orthop. 1996; 110(5): 541â546. PubMed Abstract | Publisher Full Text\n\nKatashiba S, Deguchi T, Kageyama T, et al.: The aggressive chin cup protocol (14 h/day for 2 years with excellent compliance) depends on commitment to overcorrection of the skeletal Class III malocclusion. Orthod Waves. 2006; 65(2): 57â63. Publisher Full Text\n\nLin HC, Chang HP, Chang HF: Treatment effects of occipitomental anchorage appliance of maxillary protraction combined with chincup traction in children with Class III malocclusion. J. Formos. Med. Assoc. 2007; 106(5): 380â391. PubMed Abstract | Publisher Full Text\n\nOwtad P, Park JH, Shen G, et al.: The biology of TMJ growth modification: a review. J. Dent. Res. 2013; 92(4): 315â321. Publisher Full Text\n\nPancherz H, Ruf S, Thomalske-Faubert C: Mandibular articular disk position changes during Herbst treatment: a prospective longitudinal MRI study. Am. J. Orthod. Dentofac. Orthop. 1999; 116(2): 207â214. PubMed Abstract | Publisher Full Text\n\nGökalp H, Kurt G: Magnetic resonance imaging of the condylar growth pattern and disk position after chin cup therapy: a preliminary study. Angle Orthod. 2005; 75(4): 568â575. PubMed Abstract\n\nLee H, Son WS, Kwak C, et al.: Three-dimensional changes in the temporomandibular joint after maxillary protraction in children with skeletal Class III malocclusion. J. Oral Sci. 2016; 58(4): 501â508. PubMed Abstract | Publisher Full Text\n\nGrandori F, Merlini C, Amelotti C, et al.: A mathematical model for the computation of the forces exerted by the facial orthopedic mask. Am. J. Orthod. Dentofac. Orthop. 1992; 101(5): 441â448. PubMed Abstract | Publisher Full Text\n\nde Clerck H , Nguyen T, de Paula LK , et al.: Three-dimensional assessment of mandibular and glenoid fossa changes after bone-anchored Class III intermaxillary traction. Am. J. Orthod. Dentofac. Orthop. 2012; 142(1): 25â31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHusson AH, Burhan AS, Hajeer MY, et al.: Evaluation of the Dimensional Changes in the Mandible, Condyles, and the Temporomandibular Joint Following Skeletal Class III Treatment with Chin Cup and Bonded Maxillary Bite Block Using Low-Dose Computed Tomography: A Single-center, Randomized Controlled Trial. [Dataset]. figshare. 2023. Publisher Full Text"
}
|
[
{
"id": "166186",
"date": "23 Mar 2023",
"name": "Ghassan Idris",
"expertise": [
"Reviewer Expertise Orthodontics",
"Craniofacial anomalies and Cleft Lip and Palate Children",
"Functional appliances in growing children",
"Sleep Apnoea"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEvaluation of the dimensional changes in the mandible, condyles, and the temporomandibular joint following skeletal class III treatment with chin cup and bonded maxillary bite block using low-dose computed tomography: A single-center, randomized controlled trial\nThe study is a randomized controlled trial on 38 Class III growing children. The study used the chin cup combined with maxillary bonded posterior bite planes. Volumetric and two- dimensional linear assessment were carried out using CBCT in two times. Before starting the treatment and after achieving psotive overjet in the treatment group and in 16 months in the control group.\nThe report is well written and answering an important question to fill a gap in the literature regarding the use of Chin Cup which were always debatable among orthodontists. The current repot is thorough following the CONSORT guidelines and the outcome and conclusion is related to the reported data which is satisfactory.\nOne point I feel it is a bit confusing and it needs to be clarified is the follow up times. It is clear that the follow ups were at T1 and T2 however the following sentence in the Abstract section â Low-dose CT images were acquired before (T1) and after achieving (2-4 mm) positive overjet (T2), and after 16 months apart in both groups.â Is giving the impression that there were a separate third time follow up at 16 months. Please, this sentence needs rephrasing.\nCongratulations for authors on the good work and well written report.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "166188",
"date": "28 Mar 2023",
"name": "Mohammad Khursheed Alam",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for inviting me to review this manuscript. The study aimed to evaluate the dimensional changes in the mandible and the condyles following Class III treatment in growing patients. Orthopedic forces were applied to study the post-treatment changes. An RCT design was adopted to answer the research question. A control group of untreated patients was used to filter out the changes that may occur during the observation period due to growth. The study included 38 patients who were distributed equally into the two groups (with 19 patients in each group). The method of assessment was based on low-dose CT images which were taken twice (before and after the active treatment).\nI think that this study is well-conducted and well-written, and it delivers important information regarding the dimensional changes of the condyles, glenoid fossae, and the body of the mandible after treating the chin cup in treating children with prognathic mandibles. The research question is clearly stated. The materials and methods section contains detailed information about the different steps in conducting this work. The results are displayed using good tables and figures. An in-depth discussion is given in this manuscript, and it seems the authors are well familiar with recently published papers on this topic.\nMajor points\nAfter careful reading of this paper, I don't see major points that require an author's response.\nMinor points\nThe following few points require the author's attention\nIn the Abstract section, the authors say, \"Low-dose CT images were acquired before (T1) and after achieving (2-4 mm) positive overjet (T2), and after 16 months apart in both groups\". According to my understanding from the paper, the sentence should be changed to appear as follows \"Low-dose CT images were acquired before treatment (T1) and after achieving 2 to 4 mm positive overjet with at least 16 months of active treatment (T2).\"\n\nIn the Materials and Methods section, the authors say, \"The radiological inclusion criteria were (1) mild to moderate mandibular prognathism (SNB > 80°, and 0 â€ANBâ¥4)\". Please correct this condition since the angle \"ANB\" should lay between 0 and â 4 (minus 4).\n\nIn the Materials and Methods section, the authors say that they used the ANB angle to determine Class III patients who should be enrolled in the study. If \"Witz appraisal\" was used to confirm the diagnosis, please indicate that in the text.\n\nUnder the \"Interventions\" subheading, the appliance was written \"Chin-cup\". In other places in this manuscript, it appears as \"chin cup\". Please try to mention this appliance in a standardized manner, i.e., either \"chip-cup\" or \"chin cup\", throughout the whole manuscript.\n\nIn the \"Interventional group\" subheading, please use the term \"retraction force\" instead of \"traction force\".\n\nUnder the \"Outcome measures\" subheading, the authors say that patients were seated in a supine position with their Frankfurt Horizontal (FH) plane perpendicular to the floor. This information is wrong. Their Frankfurt Horizontal plane should be parallel to the floor. Please correct.\n\nUnder the \"The mandible and condyles reconstruction\" subheading, please replace the term \"surface sizes\" with \"surface areas\".\n\nIn the \"Statistical analysis\" subsection, please add the word \"distributions\" at the end of this sentence \"Shapiro-Wilk test was used to evaluate the Noramilty of data distributions\".\n\nIn the \"Hrams\" subheading, please provide any information about symptoms related to the TMJ disorders that may have developed in the experimental group (the chin cup group). If you encountered no adverse effects on the TMJs, please declare this result in this section.\n\nIn the \"Limitations\" subheading (in the Discussion section), please add the limitation regarding the absence of any objective clinical examination of TMJ disorders after the chin cup therapy. You have already shown that there was compression in the joint spaces. Therefore, one could assume that problems may have arisen in the TMJ regions.\nOverall, the paper is well-written, and the authors should be congratulated for their great research work. The clinical message is clear from this RCT.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "166185",
"date": "11 Apr 2023",
"name": "Ahmad Hamdan",
"expertise": [
"Reviewer Expertise Orthodontics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks you for asking me to review this interesting paper.\nThis is a well designed study with an adequate sample size calculation and randomization. The methodology is sound and I am pleased that a reproducibility study was carried out, Although I would have liked to see a paired sample T test performed between repeat measurement to determine random error.\n\nStatistical analyses could benefit from multiple variable comparisons using ANOVA since changes in many of the variable are related,\nThe results are well presented and illustrated and based on the gathered data.\nDiscussion is adequate and analyses all the main results.\n\nMy main concerns are related to the high standard deviations of many of the variable which is is an indication that the data should be interpreted with caution. In addition, the influence of the bite block without chin cup is not discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "166187",
"date": "11 Apr 2023",
"name": "Rozita Hassan",
"expertise": [
"Reviewer Expertise Orthodontist"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript was overall well-written with good command of English. It is a clinical trial and utilized an advanced method of measurement technology.\nThe finding helps the clinician to understand more about the outcome of Class III malocclusion patients treated with a chin cup.\nHowever one of the radiological inclusion was 0< ANB>4 which indicated inclusive of Skeletal Class 1. Perhaps the title needs to be revised. My suggestion is to change to Class III malocclusion rather than Skeletal class III.\n\nIn a view of the chin cup type, there is a lacking of information regarding the design and company brand.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-264
|
https://f1000research.com/articles/11-773/v2
|
10 Mar 23
|
{
"type": "Systematic Review",
"title": "Epidermal growth factor outperforms placebo in the treatment of diabetic foot ulcer: a meta-analysis",
"authors": [
"Fazal Rahim",
"Xie Yan",
"Jawad Ali Shah",
"Nida Bibi",
"Zafar Ullah Khan",
"Shah Nawaz",
"Yao Ming",
"Xie Yan",
"Jawad Ali Shah",
"Nida Bibi",
"Zafar Ullah Khan",
"Shah Nawaz",
"Yao Ming"
],
"abstract": "Background: Diabetic foot ulcers (DFUs) are a life-threatening ailment caused by diabetes. Several growth factors, as well as their various combinations, have shown promising effect in aiding diabetic foot ulcer. However, contradictory or paradoxical results are often available, and debates about this issue are ongoing. Therefore, a comprehensive meta-analysis was performed to compare the efficacy and safety of epidermal growth factor (EGF) and placebo in healing diabetic foot ulcers. Methods: The database search included relevant English literature from Cochrane Library, PubMed, Google Scholar, Elsevier, and EMBASE that was published between 2009 and 2021. Inclusion criteria included type 1 and 2 diabetic patients with foot wounds focusing on complete healing rate. Exclusion criteria included combined therapy, non-human studies, reviews, and protocols. To assess the quality of each study, biases regarding random sequence generation, allocation concealment, participant and personnel blinding, outcome assessment blinding and incomplete outcome data were thoroughly identified. Results: Eight randomized control trials comprising 620 patients (337 in EGF group, 283 in placebo group), were included in this meta-analysis. EGF achieved a significantly higher complete healing rate than placebo after four weeks of treatment, with relative risk (RR): 3.04 (0.50, 18.44) and heterogeneity (Chi2 = 6.46, df = 2 (P = 0.04) I2 = 69 %). Notably, the healing frequency in the placebo group was 17%, whereas the healing frequency in the epidermal growth factor group was 34%. Likewise, after eight weeks of treatment, the relative risk and heterogeneity were RR: 2.59 (1.42, 4.72) and (Chi2 =7.92, df= 4 (p= 0.09): I2= 49%), respectively. Moreover, the risk ratio at 12 weeks was RR: 1.01 (0.42, 2.46), and heterogeneity was (Chi2 =8.55, df= 2 (p= 0.01): I2= 77%). Conclusions: Our findings indicate that EGF significantly promotes wound healing, and could be recommended as an effective and safe treatment for DFUs.",
"keywords": [
"Diabetic foot ulcer",
"epidermal growth factor",
"Placebo",
"Meta-analysis"
],
"content": "Introduction\n\nDiabetes is a metabolic syndrome, which may be due to the reduced insulin secretion, or defects in insulin function, leading to glucose deficiency, resulting in hyperglycemia.1 Following this, according to international diabetes federation 2017,2 only diabetes was responsible for four million fatalities globally. As a results, 382 million individuals were diagnosed with diabetes in 2013,3 415 million in 2015,4 425 million in 2017,2 463 million in 2019, and is projected to effect 578 million people in 2030.5 Consequently, the average growth rate in diabetes patients from 2013 to 2030 is more than 52%. Increasing tendency of morbidity and mortality is seen in patients with type 2 diabetes, which frequently leads to premature death.1\n\nThe three main types of diabetes are type 1 diabetes (T1DM), type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus (GDM). However, the onset of T2DM is more insidious accounting for roughly 90% of all cases.6 Excessive thirst, hunger, weariness, sluggishness, weight loss in type one or progressive weight gain in type two, blurred vision, and passing more urine than normal are the most typical symptoms of diabetes. Hyperglycemia, ketoacidosis, and diabetic coma are the most prevalent acute complications of diabetes.5 In addition, ageing, greater urbanization, genetics, and an obesogenic profile are the primary causes of diabetes.7 People with a family history of diabetes, for example, have a 25% increased chance of inheriting type 2 diabetes from their parents.8 Similarly, monozygotic twins are 90% more likely than heterozygotic twins to acquire T2DM later in life. Moreover, the incidence rates of T1D are also rising, contributing to the increase in diabetes prevalence worldwide.9 However, in depth knowledge for the specific cause of this rise is of paramount importance.\n\nAccording to the World Health Organisation (WHO) anticipations, diabetic foot ulcers would affect more than 19% of the worldâs adult population by 2030.10 Diabetic foot ulcers (DFUs) are wounds in the dermis (the skinâs deep blood vessels and collagen inner layer) that appear below the ankles of diabetic patients.11 It is estimated that DFUs affect 9.1 to 26.1 million diabetic patients each year across the globe.12 Diabetic foot ulcers were found to be prevalent in 6.3% of the worldâs population, with North America (13%) having the highest prevalence and Oceania having the lowest (3%). In Asia, Europe, and Africa, the prevalence was 5.5%, 5.1%, and 7.2%, respectively.13 Indeed, diabetes mellitus is one of the most common causes of non-traumatic lower extremity amputation. Approximately 20% of diabetic foot infections that are mild to severe result in amputation.14 The risk of deaths in DFU individuals dramatically raised by 2.5 times when compared to non-DFU patients.2,12 Insufficient blood circulation due to malfunction of circulatory system significantly increase the incidence of diabetic foot ulcers. Therefore, to treat DFU patients more effectively, it is extremely important to develop a cutting-edge treatment approach.\n\nSeveral growth factors, including platelet-derived growth factor, fibroblast growth factor, epidermal growth factor (EGF), and peripheral blood mononuclear cells, have showed promise in accelerating ulcer healing in various combinations.15â17 Inflammation, proliferation, and remodeling are the three stages of wound healing, each of which necessitates the coordination and integration of delicate and complicated biological activities. Chemotaxis, cell proliferation, extracellular matrix deposition, angiogenesis, and tissue reconstruction are all stimulated by the growth factors involved in those biological activities. Several published literature have evaluated the curative effect of topical EGF and placebo on healing diabetic foot ulcers, but there are always contradictions in the evidence to distinguish the true therapeutic effect and safety issues of EGF and placebo in the treatment of DFUs.18,19 Therefore, a comprehensive meta-analysis was conducted to evaluate the efficacy and safety of EGF and placebo on healing diabetic foot ulcers. The application of EGF, according to our hypothesis, outperforms placebo in facilitating the healing process of DFUs.\n\n\nMethods\n\nThe meta-analysis was reported according to the preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement.20,29\n\nThe search for relevant English literature published between 2009 to 2021 was performed in Cochrane Library, PubMed, EMBASE, and Google Scholar. The specific key terms used in this study includes, Epidermal Growth Factor (EGF), Placebo, Diabetic Foot Ulcer, TDM1 and TDM2. Studies from the reference list were also incorporated in order to find more relevant material. Articles were located and checked on a variety of levels, including title, abstract, and full-text. Final papers that met the inclusion criteria retrieved and included in the study, while those that did not were initially excluded. The full texts of potentially eligible studies were obtained and double-screened for eligibility by two potentials reviewers (Figure 1). Disagreements were resolved through discussion.\n\nInclusion criteria for selecting studies included:\n\ni) Type 1 and 2 diabetic patients with foot wounds.\n\nii) Literature mainly focused on complete healing time.\n\niii) Any study design including double-blind, placebo-controlled trial, randomized control trails, retrospective study, prospective study.\n\niv) Studies comparing epidermal growth factors and placebo in the treatment of diabetic foot ulcers.\n\nExclusion criteria comprised:\n\ni) Combined therapy\n\nii) Studies with no reported sample size\n\niii) Single study of epidermal growth factor or placebo treatment.\n\niv) Non-human studies, reviews, protocols, and trials.\n\nThe following data were extracted from the eligible studies: initial authorâs name, year of publication, location, study time (start-to-end date), number of patients, study design, patient characteristics (average age, sex, etc.), and treatment duration. Authors of articles with insufficient data were contacted for details. Papers were not included in the meta-analysis if the contacted author did not provide the required data. Two independent reviewers extracted the required data.\n\nTo assess the quality of each study, the Cochrane risk, a biased evaluation tool in the Review Manager 5.4 programme for RCTs, and the Newcastle-Ottawa Scale (NOS) for cohort studies were employed. We looked at random sequence generation (selection bias), allocation concealment (selection bias), participant and personnel blinding (performance bias), outcome assessment blinding (detection bias), incomplete outcome data (attrition bias), and other sources of bias. In each domain, studies were classified as having a high (red), unclear (yellow), or low (green) risk of bias. The RevMan software regenerated the risk of bias summary table and graph (version 5.4 Copenhagen: The Nordic Cochrane Center, the Cochrane Collaboration, 2014, Denmark). Furthermore, we created a funnel plot to see if there was any publishing bias.\n\nReview Manager 5.4 were used to compile all of the data (RevMan, the Cochrane cooperation, Oxford, UK, RRID:SCR_003581). For this meta-analysis, the heterogeneity between studies was assessed by Cochran (Q) and I2 statistics, which expresses the percentage of variation between studies. I2 was used to evaluate inter study heterogeneity. An I2 value higher than 50% was considered to have statistically significant heterogeneity. We presented dichotomous outcomes as risk ratios (RRs) with their corresponding 95% confidence intervals (CIs). Fixed or random effect techniques are used in meta-analysis, depending on the degree of heterogeneity. The random effect approach is used to combine results when significant heterogeneity is identified in the data, whereas the fixed effect method is used when significant heterogeneity is not detected.\n\n\nResults\n\nIn a systematic search of various electronic databases, a total of 993 articles were identified (EMBASE 189, Google Scholar 293, Cochrane Library 123, Elsevier 192, and PubMed 196). Following an initial screening, 308 studies were ruled out due to their titles. Following that, the remaining 685 articles were carefully screened, with 491 studies being excluded based on full text or abstract, including combination therapies (n = 117), no comparison between epidermal growth factor and placebo (n = 96), non-diabetic (n = 82), non-human studies (n = 45), basic science (n = 78), and no clinical trials (n = 73). The remaining 194 papers were evaluated more thoroughly, and 186 were eliminated due to data gaps (n = 27), duplicate studies (n = 68), content of studies without the desired outcome (n = 69), and wound type mismatch (n = 22). Finally, eight randomized controlled studies were included in the meta-analysis. Figure 1 shows the identified and retrieved articles in the study.\n\nIn total, eight randomized control trials (RCTs) that involved a total of 620 patients (337 in the EGF group) and 283 in the placebo group, were included. Except one study from Mexico and another from Cuba, the majority of the studies were from Asia. In these studies, patients received either EGF or placebo intervention, in addition to standard diabetic foot management. The EGF and placebo treatments were administrated by intralesional injection or topical application. All of the patients had type 1 or type 2 diabetes with DFUs. The patientâs ages ranged from 20 to 75 years old. The majority of studies had a follow-up period of 4 to 14 weeks. Table 1 shows the basic characteristics of the included studies.\n\nFigures 2 and 3 show more information on the risk of bias assessment. The quality evaluation was carried out on a total of eight studies that were included in the qualitative analysis, with the results indicating that the risk of bias was mostly low and unclear. Our risk of bias assessment reported a reasonable quality after adapting the Cochrane Risk of Bias Tool to the Agency for Healthcare Research and Quality (AHQR) standard. The funnel plot exhibited a clear symmetric trend, confirming the absence of publishing bias (Figure 4).\n\nStatistical analysis results are shown in Figure 5. Three publications including 85 patients were divided into two groups. The epidermal growth factor group recovered significantly faster than the placebo group after four weeks of treatment. For example, with four-week treatment, the healing frequency in the placebo group was 17%, whereas the healing frequency in the epidermal growth factor group was 34%. Furthermore, there is a substantial difference in the healing rate between the epidermal growth factor and placebo groups after four weeks of treatment. Figure 5 shows a significant difference in risk ratio (RR: 3.04, [95 % CI: 0.50, 18.44] I2 = 69%) and heterogeneity (Chi2 = 6.46, df = 2 (P = 0.04) I2 = 69%).\n\nAbbreviations; CI, confidence interval; df, degrees of freedom; EGF, epidermal growth factor.\n\nThe eight-week healing rate of epidermal growth factor and the placebo group is compared in five articles. There were 123 patients in the epidermal growth factor group and 125 in the placebo group. After eight weeks of therapy, 79% of patients were recovered with the application of epidermal growth factor, while 25% were recovered in the placebo group, indicating a significant difference (Figure 6). The proportion of complete ulcer healing with EGF was significantly higher than that of placebo (RR: 2.59, [95% CI: 1.42, 4.72] I2 = 49%) and (Chi2 = 7.92, df = 4 (p = 0.09): I2 = 49%).\n\nAbbreviations; CI, confidence interval; df, degrees of freedom; EGF, epidermal growth factor.\n\nA total of three papers compared the healing rate of EGF and placebo groups after twelve weeks of therapy. The EGF group received 134 patients, while the placebo group received 135. There was a clear difference between the epidermal growth factor and placebo groups after twelve weeks of treatment (Figure 7). The epidermal growth factor group had a healing rate of 71%, while the placebo group had a healing rate of 58%. Furthermore, a significant difference in risk ratio and heterogeneity were noted, with risk ratio (RR: 1.01, [95% CI: 0.42, 2.46] I2 = 77%) and heterogeneity (Chi2 = 8.55, df = 2 (p = 0.01): I2 = 77%).\n\nAbbreviations; CI, confidence interval; df, degrees of freedom; EGF, Epidermal growth factor.\n\nStatistical analysis results for complete healing rate of diabetic foot ulcers in patients treated with EGF versus placebo is shown in Figure 8. The complete healing rate study includes a total of eight publications, with 620 patients demonstrating a complete healing rate; 337 in the EGF group and 283 in the placebo group. For instance, in the epidermal growth factor group a total of 245 (out of 337) healing events occurs, while in placebo group a total of 138 (283) healing events occurs. The risk ratio and heterogeneity were (RR: 1.50, [95% CI: 1.32, 1.71] I2 = 21%) and heterogeneity (Chi2 = 8.81, df = 7 (p = 0.27): I2 = 21%). Overall, the complete healing rate of epidermal growth factor was significantly higher than that of placebo and it could be used as a recommended first line therapy for treatment of DFUs (Figure 8).\n\nAbbreviations; CI, confidence interval; df, degrees of freedom; EGF, epidermal growth factor.\n\n\nDiscussion\n\nThis study was carried out to clarify the efficacy and safety of EGF and placebo in the treatment of diabetic foot ulcer. A total of 620 diabetic patients with foot ulcers were included in the meta-analysis. Several meta-analyses have been published in the last decade21â23; however, previously published studies frequently share some common limitations. For example, the quality of risk-of-bias was insufficient because of the unknown performance of bias since the patients were not blinded, which could lead to an overestimation of the studyâs quality. Another disadvantage is that most studies do not contain any relevant literature at all. Furthermore, past research has employed protocol analysis,22 which has the potential to disrupt the baseline character balance and overestimate treatment outcomes. Therefore, taking all of the inherent limitations into account, a meta-analysis was undertaken based on eight trials that included data from various nations aiming to compare EGF vs placebo alone. The EGF group included 337 patients, while the placebo group includes 283 patients. Our results indicated that the use of EGF significantly improves the healing rate of DFUs relative to placebo group. For instance, the EGF group shows a complete healing rate of 71% while the placebo group shows a complete healing rate of 58.7%. Our findings are similar to those of Quoc Van Phu Bui et al.,22 who discovered that epidermal growth factor therapy is superior over placebo. Furthermore, Viswanathan et al.,24 determined a substantial difference between epidermal growth factors and placebo therapy. For example, the EGF group had a complete healing rate of 78%, whilst the placebo group had a complete healing rate of 52%. The most positive conclusion in our study is that EGF improves foot ulcer healing considerably. As a result, as compared to placebo, EGF therapy results in a faster recovery of the wound. The significance of growth factors in wound repair could be one explanation for the remarkable therapeutic impact. EGF promotes epidermal cell proliferation by stimulating glycolysis, mitosis, and protein synthesis.25 By causing inflammatory cells to relocate to ulcer sites, EGF can enhance the wound microenvironment and tissue nutrition. Furthermore, Thambi Durai David et al.26 noted that patients treated with EGF had a substantially higher rate of complete healing rates than those treated with placebo i.e. the percentage of total recovery in the EGF and placebo groups was 71.2% and 48.9%, respectively (Figure 8). Over the course of four weeks, a significant difference was found in cure rates between the EGF and placebo groups, ranging from 3-92% to 0-65%, respectively. Our obtained results are compatible with the findings of the previous meta-analysis.21 For example, Yang et al.,21 found that, EGF therapy outperformed placebo and the healing rate was 23% in the EGF group and 10% in the placebo group (Figure 6). Consequently, after eight weeks of therapy, there was a substantial difference between the EGF and placebo groups, with healing rates of 16-92 % and 0-25%, respectively. Following this in a recent meta-analysis, Kumar et al.,27 found a significant difference between the EGF group and the placebo group, i.e. in the EGF group, complete wound healing was 76%, compared to 20% in the placebo group. Moreover, the average recovery time for totally healed ulcers was eleven weeks (Figure 7). However, wound healing in the EGF and placebo groups differed significantly after 12 weeks of therapy. The EGF group had a healing rate of 20-73%, while the placebo group had a healing rate of 11-52%. According to previous research by Yang et al.,28 EGF is far superior to the placebo group, with a complete cure rate of 23.5-95.3% in EGF and 0 to 52.1% in the placebo group. Overall, our findings provide meaningful information to current treatment scenarios of DFUs. As a result, based on current evidence, EGF administration to DFUs is considered as effective and safe.\n\nThe most important strengths of our meta-analysis are that; first to our knowledge with eight RCTs this is the most comprehensive meta-analysis on comparing EGF and placebo for DFUs. We also looked for suitable references and emailed the authors for the missing information. Our meta-analysis includes several current clinical studies, which provided more evidence than the prior meta-analysis. This is significant because the number of trials comparing EGF to placebo in the treatment of DFU is currently limited. However, even though publication bias analysis was performed, there are still some potential biases with this review. Some of the literatures included were of poor quality. Although the authors reported that their studies were randomized, the random sequences and blind details were not described in the original articles. The origin of the works did not correspond to a homogeneous recruitment. The economic analysis is important for patients with long diabetic history, especially in developing countries. However, most of the trials didnât provide cost-effectiveness data. The amount of data on adverse effects was also limited, and hence we could not elicit the most common side effect experienced as a result of EGF and placebo treatment.\n\n\nConclusion\n\nCompared to placebo therapy, EGF significantly accelerate the healing of diabetic foot ulcers at 4-12 weeks of treatment. The EGF has the potential to improve ulcer rehabilitation and speed up wound healing. This conclusion, however, should be approached with caution. More well-designed clinical trials in different populations with long follow-up time are required to further examine the topical EGF therapy in management of diabetic foot ulcer in the future.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReporting guidelines\n\nFigshare: PRISMA checklist for âEpidermal growth factor outperforms placebo in the treatment of diabetic foot ulcer: a meta-analysisâ. https://doi.org/10.6084/m9.figshare.19928645.29\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nUnuofin JO, Lebelo SL: Antioxidant Effects and mechanisms of medicinal plants and their bioactive compounds for the prevention and treatment of type 2 diabetes: An updated review. Oxidative Med. Cell. Longev. 2020; 2020: 1â36. PubMed Abstract | Publisher Full Text\n\nInternational Diabetes Federation: IDF Diabetes Atlas. 8th ed.Brussels, Belgium: International Diabetes Federation; 2017. Reference Source\n\nInternational Diabetes Federation: IDF Diabetes Atlas. 6th ed.Brussels, Belgium: International Diabetes Federation; 2013. Reference Source\n\nInternational Diabetes Federation: IDF Diabetes Atlas. 7th ed.Brussels, Belgium: International Diabetes Federation; 2015. Reference Source\n\nSaeedi P, Petersohn I, Salpea P, et al.: Global and Regional Diabetes Prevalence Estimates for 2019 and Projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9th Edition. Diabetes Res. Clin. Pract. 2019; 157: 107843. PubMed Abstract | Publisher Full Text\n\nDavis IC, Ahmadizadeh I, Randell J, et al.: Understanding the impact of hypoglycemia on the cardiovascular system. Expert. Rev. Endocrinol. Metab. 2017; 12(1): 21â33. PubMed Abstract | Publisher Full Text\n\nAlotaibi A, Perry L, Gholizadeh L, et al.: Incidence and prevalence rates of diabetes mellitus in Saudi Arabia: an overview. J. Epidemiol. Glob. Health. 2017; 7(4): 211â218. PubMed Abstract | Publisher Full Text\n\nKlein BE, Klein R, Moss SE, et al.: Parental history of diabetes in a population-based study. Diabetes Care. 1996; 19(8): 827â830. PubMed Abstract | Publisher Full Text\n\nPatterson CC, Dahlquist GG, Gyurus E, et al.: EURODIAB Study Group. Incidence trends for childhood type 1 diabetes in Europe during 1989â2003 and predicted new cases 2005â20: a multicentre prospective registration study. Lancet. 2009; 373(9680): 2027â2033. PubMed Abstract | Publisher Full Text\n\nGrennan D: Diabetic foot ulcers. JAMA. 2019; 321(1): 114. Publisher Full Text\n\nArmstrong DG, Boulton AJM, Bus SA: Diabetic foot ulcers and their recurrence. N. Engl. J. Med. 2017; 376(24): 2367â2375. Publisher Full Text\n\nWalsh JW, Hoffstad OJ, Sullivan MO, et al.: Association of diabetic foot ulcer and death in a population-based cohort from the United Kingdom. Diabet. Med. 2016; 33(11): 1493â1498. PubMed Abstract | Publisher Full Text\n\nZhang P, Lu J, Jing Y, et al.: Global epidemiology of diabetic foot ulceration: A review and meta-analysis. Ann. Med. 2017; 49(2): 106â116. PubMed Abstract | Publisher Full Text\n\nInternational Diabetes Federation the global burden: IDF diabetes atlas.5th ed.2012. Reference Source\n\nOnodera R, Teramukai S, Tanaka S, et al.: Bone marrow mononuclear cells versus G-CSF-mobilized peripheral blood mononuclear cells for treatment of lower limb ASO: pooled analysis for long-term prognosis. Bone Marrow Transplant. 2011; 46(2): 278â284. PubMed Abstract | Publisher Full Text\n\nChoi SM, Ryu HA, Lee KM, et al.: Development of Stabilized Growth Factor-Loaded Hyaluronatee Collagen Dressing (HCD) matrix for impaired wound healing. Biomater Res. 2016; 20: 9. PubMed Abstract | Publisher Full Text\n\nLee CH, Liu KS, Chang SH, et al.: Promoting diabetic wound therapy using biodegradable rhPDGF-Loaded nanofibrous membranes: CONSORT-compliant article. Medicine. 2015; 94(47): e1873. PubMed Abstract | Publisher Full Text\n\nHong JP, Jung HD, Kim YW: Recombinant human epidermal growth factor (EGF) to enhance healing for diabetic foot ulcers. Ann. Plast. Surg. 2006; 56(4): 394â398. PubMed Abstract | Publisher Full Text\n\nPersiani F, Paolini A, Camilli D, et al.: Peripheral Blood Mononuclear Cells Therapy for Treatment of Lower Limb Ischemia in Diabetic Patients: a Single-Center Experience. Ann. Vasc. Surg. 2018; 53: 190â196. Publisher Full Text\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration. BMJ. 2009; 339: b2700. PubMed Abstract | Publisher Full Text\n\nYang S, Geng Z, Ma K, et al.: Efficacy of topical recombinant human epidermal growth factor for treatment of diabetic foot ulcer: a systematic review and meta-analysis. Int. J. Low Extrem. Wounds. 2016; 15(2): 120â125. PubMed Abstract | Publisher Full Text\n\nThien Quoc B, Quoc VPB, Nemeth D, et al.: Epidermal growth factor is effective in the treatment of diabetic foot ulcers: meta-analysis and systematic review. Int. J. Environ. Res. Public Health. 2019; 16(14): 2584.\n\nYang Q, Zhang Y, Yin H, et al.: Topical recombinant human epidermal growth factor for diabetic foot ulcers: a met analysis of randomized controlled clinical trials. Ann. Vasc. Surg. 2020; 62: 442â451. PubMed Abstract | Publisher Full Text\n\nViswanathan V, Juttada U, Babu M: Efficacy of Recombinant Human Epidermal Growth Factor (Regen-D 150) in Healing Diabetic Foot Ulcers: A Hospital-Based Randomized Controlled Trial. Int. J. Low Extrem. Wounds. 2019; 19(2): 158â164. Publisher Full Text\n\nMohan VK: Recombinant human epidermal growth factor (REGEN-D 150): effect on healing of diabetic foot ulcers. Diabetes Res. Clin. Pract. 2007; 78(3): 405â411. PubMed Abstract | Publisher Full Text\n\nThambi DD, Jayalal JA, Selwyn JK: The Role of Epidermal Growth Factor Cream in Healing of Diabetic Foot Ulcer - Comparative Analytical Study in South India. Int. J. Health Sci. Res. 2019; 8(6): 2249â9571. Publisher Full Text\n\nKumar A, Sinha S, Khandelwal B: Efficacy of topical recombinant epidermal growth factor as compared to Povidone Iodine on chronic diabetic foot ulcers. Int. J. Health Clin. Res. 2020; 3(4): 82â88.\n\nQi Y, Zhang Y, Yin H, et al.: Topical Recombinant Human Epidermal Growth Factor for Diabetic Foot Ulcers: A Meta-Analysis of Randomized Controlled Clinical Trials.2020; 62: 442â451. Publisher Full Text\n\nFazal R, Shah JA: PRISMA_2020_checklist.docx. figshare.2022. Online resource. Publisher Full Text"
}
|
[
{
"id": "202888",
"date": "18 Sep 2023",
"name": "Chin Chai Yee",
"expertise": [
"Reviewer Expertise My area of research is lies on biomaterial development and investigating the mechanism of the wound healing effects using bioactive compounds or synthetic drugs."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe overall conclusions drawn in the review appear to be generally supported by the results presented in the meta-analysis. The review provides a comprehensive analysis of eight randomized controlled trials (RCTs) by comparing the efficacy and safety of EGF (Epidermal Growth Factor) versus placebo in the treatment of diabetic foot ulcers (DFUs). Here are some constructive comments based on the results and discussion:\nThe review rightly highlights its strengths, including its comprehensive approach with eight RCTs, the efforts made to gather missing information, and the inclusion of recent clinical studies. These strengths enhance the credibility of the meta-analysis and provide more robust evidence compared to prior studies.\n\nThe review effectively draws comparisons with previous meta-analyses and individual studies, showing that its findings align with existing evidence. This strengthens the case for the efficacy of EGF in DFU treatment.\n\nThe review acknowledges several limitations, such as the lack of detailed information on randomization and blinding in some studies, heterogeneity in the recruitment criteria, and the absence of cost-effectiveness and adverse effects data. Recognizing these limitations is important as it highlights potential sources of bias and areas where future research can improve.\n\nWhile the review highlights that EGF therapy significantly improves DFU healing compared to placebo, it should emphasize the clinical significance of this improvement. What does it mean for patients in terms of healing time and overall outcomes? Providing more context to the results would enhance the understanding of their practical implications.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
},
{
"id": "211405",
"date": "19 Oct 2023",
"name": "Poonam Kushwaha",
"expertise": [
"Reviewer Expertise Exploration of nanotechnology in enhanced dermal drug delivery."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis meta-analysis provides valuable insights into the effectiveness of epidermal growth factor (EGF) in the treatment of diabetic foot ulcers. The comprehensive analysis of eight randomized controlled trials adds to the growing evidence supporting EGF as a promising intervention for diabetic foot ulcers. The study's findings align with previous research, highlighting the significant impact of EGF on accelerating wound healing.\n\nThe authors have done a commendable job in conducting a rigorous analysis and assessing the risk of bias in the included studies. The quality assessment and the absence of publication bias, as indicated by the funnel plot, contribute to the credibility of the findings. This strengthens the case for EGF as a recommended treatment for diabetic foot ulcers.\n\nDiabetic foot ulcers pose a substantial health risk and economic burden, particularly in the face of the rising prevalence of diabetes worldwide. This meta-analysis underscores the urgency of developing effective treatments for diabetic foot ulcers and the potential role of EGF in reducing the associated morbidity and mortality.\n\nThe study's emphasis on the need for cost-effectiveness data is significant, especially for resource-constrained settings. Future research could delve into the economic implications of EGF treatment in different healthcare systems, shedding light on its affordability and accessibility to a wider population of diabetic patients.\n\nOne limitation of this meta-analysis is the lack of comprehensive data on adverse effects. Understanding the safety profile of EGF in the context of diabetic foot ulcer treatment is crucial. Future studies should prioritize reporting adverse events to provide a more complete picture of the intervention's overall risk-benefit profile.\n\nThe consistent findings of this meta-analysis reinforce the importance of growth factors in wound healing, and EGF's role in promoting cell proliferation and enhancing the wound microenvironment. These biological mechanisms provide a scientific basis for EGF's efficacy in diabetic foot ulcer treatment.\nOverall, this study contributes significantly to the field of diabetic foot ulcer management. The evidence presented here supports the use of epidermal growth factor as an effective and safe treatment option. This meta-analysis is a valuable resource for clinicians and researchers working to improve the care and outcomes of diabetic patients with foot ulcers.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": []
}
] | 2
|
https://f1000research.com/articles/11-773
|
https://f1000research.com/articles/12-259/v1
|
10 Mar 23
|
{
"type": "Software Tool Article",
"title": "PyRadGUI: A GUI based radiomics extractor software",
"authors": [
"Umesh B. Sherkhane",
"Ashish Kumar Jha",
"Sneha Mithun",
"Vinay Jaiswar",
"Alberto Traverso",
"Leonard Wee",
"Venkatesh Rangarajan",
"Andre Dekker",
"Umesh B. Sherkhane",
"Sneha Mithun",
"Vinay Jaiswar",
"Alberto Traverso",
"Leonard Wee",
"Venkatesh Rangarajan",
"Andre Dekker"
],
"abstract": "Radiomics is the method of extracting high throughput mathematical and statistical features from medical images. These features have the potential to characterize the underlying pathology of the disease that is inappreciable to a trained human eye. There are several open-source and licensed tools to extract radiomic features such as pyradiomics, LIFEx, TexRAD, and RaCat. Although pyradiomics is a widely used radiomics package by researchers, this software is not very user-friendly and can be run using a command line. We have developed and validated the GUI tool, PyRadGUI to make the radiomics software easy to operate. This software adheres to IBSI radiomic feature definition and implements the radiomic pipeline in batch processing to extract radiomic features from multiple patientâs data and stores it in a comma separated value (CSV). We validated PyRadGUI software with the existing pyradiomic pipeline.",
"keywords": [
"Graphical User Interface (GUI)",
"machine learning",
"radiomics",
"pyradiomics",
"plastimatch",
"3DSlicer"
],
"content": "Instruction\n\nAs per the World health organization (WHO), cancer is the second leading cause of death worldwide. Globally one out of six deaths are caused by cancer alone which amounts to 9.6 million deaths in 2018 (WHO-Cancer, 2022; Ferlay et al., 2019). Treatment of cancer has always remained a challenging task for the oncology community. Although earlier diagnosis and treatment are often associated with a better outcome of treatment, at the same time selection of appropriate patients for appropriate treatment is important (Stewart et al., 2018). Considering the complexity of this disease and treatment, oncology is gradually moving towards personalized medicine (Agyeman and Ofori-Asenso, 2015). A pathological test is considered a confirmatory test for cancer. Imaging tests like Computed Tomography (CT)/Positron Emission Tomography (PET)/Magnetic Resonance Imaging (MRI) also play an important role in diagnosis, treatment planning, and follow-up of the disease (Caers et al., 2014). In the last several years, the role of imaging and various advanced tests like immunohistochemistry (IHC), polymerase chain reaction (PCR), and gene sequencing has been increasing gradually in personalizing the treatment. Similarly, the role of artificial intelligence (AI) and radiomics has also witnessed a surge in oncology research over the last few years. Radiomics has been identified as an area of research to develop imaging biomarkers for the personalized treatment of cancer (Lambin et al., 2012; Aerts et al., 2014; Goodwin et al., 2017). Radiomics is a method to extract high throughput data from medical images. These features have the potential to uncover disease characteristics that are not appreciated by the expert radiologist or imaging personnel through visual interpretation (Yip et al., 2017). As radiomic features are extracted directly from medical images it provides a non-invasive method for tumor characterization as demonstrated by various researchers in the past (Lambin et al., 2012; Goodwin et al., 2017; Aerts et al., 2014). Researchers have shown the role of radiomics as a clinical predictor helping in advanced cancer care as personalized medicine in cancer (Haider et al., 2020). Apart from its role in precision diagnoses and characterization of a tumor, the role of radiomics has also been demonstrated in treatment planning (Limkin et al., 2017).\n\nSeveral open-source and licensed software packages for radiomic extraction, like IBEX, RaCaT, CaPTK, LifeX or CGITA, Pyradiomics, and TexRad have been developed and used by several researchers in the past (Pfaehler et al.,2019; Nioche et al., 2018; Zhang et al., 2015; Davatzikos et al., 2018; Johnson, 2015; Haralick et al., 1973).\n\nThe main challenges with these software packages are the complexity of radiomic extraction and the lack of standardized feature extraction from this software. The mathematical feature definitions have been provided image biomarker standardization initiative (IBSI) to standardize the radiomic extraction. It has also provided the phantom data sets with radiomic feature values (Zwanenburg, 2017; Zwanenburg et al., 2016). Although IBSI standard is widely known, only a very few radiomic softwares have standardized the entire radiomic pipeline for feature extraction. Furthermore, the majority of these radiomic extractors have operability issues and not all defined features are extracted as defined by IBSI. Pyradiomics is a widely-used open source radiomics package and adheres to the IBSI standards, but it is not user-friendly. For instance, Pyradiomics is run on a command prompt and customization is technically demanding. Hence, the use of this software is cumbersome and technically demanding for clinical doctors or scientists.\n\nIn this study, we have developed and validated a graphical user interface (GUI) based radiomic extraction software using the pyradiomics package. This software adheres to all features defined by the IBSI.\n\n\nMethods\n\nThis work is part of the Big Imaging data approach for Oncology in a Netherlands India Collaboration (BIONIC) and âpersonal health Train for radiation oncology in India and the Netherlandsâ (TRAIN) project is approved by the IEC of the hospital as a retrospective study. This software (PyRadGUI) was developed using Python open-source software on Windows systems.\n\nThis study was approved by the hospital Institutional Ethics Committee (Institutional Ethics Committee-I, Tata Memorial Centre [IEC, TMC], Mumbai, India Approval Number: 1905; dated: October 05, 2017) as a retrospective study, with waivers of informed consent from involved patients as per IEC policy of our hospital by the same Ethics Committee.\n\nPyRadGUI front end was developed by using the open-source software Python 3.6.5 (Python, 2022) and the Python tk8.6 (Python tk, 2022) module was used for the development of the graphical user interface (GUI). The open source Plastimatch package 1.8.0 (Plastimatch, 2022) was used for Digital Imaging and Communications in Medicine (DICOM) to Nearly Raw Raster Data (NRRD) conversion of imaging data and the radiomic package in python; Pyradiomics 3.0 (Griethuysen et al., 2017) was used for radiomic feature extraction. This software loads DICOM images and region of interest (ROI) from a specific folder on the computer. First, it converts the image and ROI in NRRD format using Plastimatch 1.8.0, subsequently, it extracts radiomic features from the image and finally stores the output in comma separated values (CSV) format in an output folder on the computer. The details of the software are described below (Figure 1).\n\nThe user has to select the input folder containing the Digital Imaging and Communications in Medicine (DICOM) image and radiotherapy (RT) structure files. We can select the output folder and also customize the feature extraction process by changing values in the â.yamlâ file.\n\nTo start PyRadGUI, GUI_batch_radiomics.py is run on the command prompt. GUI, shown in Figure 2, has been divided into two parts, i.e., left and right containers. The left container contains three tabs that are used for customization and radiomic extraction and the right container displays the process and error if any.\n\nAfter starting the program, the user must select an input folder containing multiple patient images and a Radio therapy (RT) structure in DICOM format. Next, the user must select an output folder and then change the feature extraction settings from the settings tab.\n\n\n\n1. Selection of image type (Original, Laplace of Gaussian, and Wavelet): To extract features on the original image, we can select the image type as the original. If feature extraction has to be done on the transformed image, then we can select either LoG (Laplace of Gaussian) or Wavelet or both.\n\n2. Selection of feature types: We have to specify the type of features we want to extract. The default includes all 1093 features from all image types.\n\n3. Bin width selection: In the customization window one can select bin width as per the user requirement and the default bin width is 25. We have used 25 bin widths for CT and MRI and 0.5 bin width for PET radiomic extraction.\n\n4. Sigma value for LoG features: In the customization window one can select the sigma value for LoG features. We have selected default 1-, 2-, and 3-mm sigma values for radiomic feature extraction.\n\n5. Selection of resampled pixel spacing: In the customization window one can select pixel spacing as desired by the user for radiomic extraction. We have used the default 2Ã2Ã2 cubic mm pixel spacing.\n\nAfter the settings have been customized, radiomics extraction can be started by clicking on the radiomics extraction button. The batch extraction starts by loading the DICOM folder and calls plastimatch. Plastimatch takes the reference CT folder and converts the input DICOM to NRRD (nearly raw raster data) format. It converts the image to image.nrrd and mask to mask.nrrd. Pyradiomics takes the converted nrrd image, nrrd mask, and the settings specified by the user and then extracts the radiomics features and writes the output in CSV format in the selected output folder. In this output, CSV file columns represent radiomic features and rows represent individual patients. The first column of the file is the patient identification number. This CSV file can be used for various analyses. Status of the running of the process, success, failures, and error reports are displayed in the right container in GUI, and processing reports are stored in the output folder as log files.\n\nWe have developed and tested our software on two computer systems (machines) using various software packages. The details of system information and software packages are shown in Table 1.\n\nIn total 50 non-small cell lung carcinomas (NSCLC) patientsâ PET/CT data with delineation and 20 chondrosarcoma patientsâ MRI data with delineation who were imaged between 2014 to 2017 were used for validation and performance testing of this software. The patientâs demographic data is shown in Table 2. Images from 100 NSLC patients were utilized to test the batch processing.\n\nSoftware validation was performed by comparing Radiomic feature value extracted using PyRadGUI Workflow (PrGW) and the two reference workflows i.e., Reference Workflow1(RW1) (3DSlicer +Pyradiomics) and Workflow2 (RW2) (Plastimatch +Pyradiomics). The same version of the Pyradiomic package and Plastimatch was used for all the workflows. For the comparison, CT imaging data from 10 patients, PET imaging data from five patients, and MRI imaging data from five patients were all employed. PyRadGUI validation algorithm workflows are shown in Figure 3.\n\nReference Workflow1 (RW1): Individual patientâs DICOM image and ROI were loaded in 3D slicer 3.0. The first appropriateness of tumor delineation was checked by an experienced imaging physicist (15 years of experience). Image and ROI were converted in NRRD format and the image was saved as an image.nrrd and ROI as label.nrrd in the patient's image folder. Again, the NRRD image and label were loaded in 3D Slicer, and appropriateness on tumor delineation was checked by the same physicist. Subsequently, the radiomic feature was extracted and saved in CSV format using the pyradiomic package on the command prompt. The same process was repeated for all 20 patientsâ data.\n\nReference Workflow2 (RW2): DICOM image and RTStructure were converted in NRRD format using Plastimatch 1.8.0 and stored similarly as it is done in Workflow1. Subsequently, radiomic features were extracted and stored in CSV format similarly as it is done in workflow1. The algorithm used for the manual extraction of radiomic features is shown in Figure 3. All the patientâs radiomic feature data was arranged similarly as it was arranged in the automated extraction of radiomic feature data as described in the automatic extraction section.\n\nOur PyRadGUI workflow was compared with manual Reference Workflow1 and Reference Workflow2 as shown in Figure 3. Interclass correlation (ICC) was calculated for all 1093 radiomic features to compare PyRadGUI workflow and Reference Workflow1 using python code. As we were expecting the same result in PyRadGUI Workflow and Reference Workflow2, we used the EXACT function of Microsoft Excel 2007 software to compare both the data sets. The EXACT function compares two strings for all the characters, if all the characters are the same in both the strings it gives true as an output otherwise false.\n\nThe performance of the software was tested for batch processing on the two machines mentioned in Table 1. CT and PET DICOM data were used in the batches of 10, 20, 50, and MRI DICOM data were used in the batches of 10, and 20. The total time required for individual batch processing was recorded.\n\n\nResults\n\nWe successfully installed and ran the software on the two computers mentioned in Table 1. All the customizations for radiomic extraction work well on multiple trials. With this software, we were able to perform radiomic extraction by batch processing of up to 100 patientsâ CT data on both computers.\n\nThe ICC value for the comparison of PrGW and RW1 was ICC =0.978 (range: 0.9612-1.0) across all the modalities. The ICC value for the comparison of PrGW and RW1 is shown in Table 3. All the values (for 1093 radiomic features) in the validation steps for CT, PET, and MRI features for PrGW and RW2 were TRUE, which shows that we were able to extract the same values for all the features for the same data across all the modalities by using this software as confirmed by the manual process.\n\nOn both machines the batch processing performance was found to be satisfactory. The details of run time are shown in Table 4. Various imaging modalities were used to test the PyRadGUI tool.\n\n\nDiscussion\n\nSeveral licensed and open-source software are available for radiomic extraction, which can extract radiomic features from two- or three-dimensional medical images (Gotz et al., 2019; Szczypinski et al., 2009; Zhang et al., 2015; Apte et al., 2018).\n\nShi et al. (2019) have developed an Ontology-guided radiomics analysis workflow (O-RAW) using the Pyradiomics package and SITK Python package, which is also able to extract radiomics features from DICOM images and RTSTRUCT in batch processing and store it in resource description framework (RDF) triple store. Another radiomic extraction software, TexRAD, is a licensed package GUI-based system. It has been used by many researchers for radiomic extraction. TexRAD is unable to do the batch processing and it can handle one patientâs data at a time (Gotz et al., 2019). This software has capabilities to display and review the image and delineate tumors manually, but another drawback of this software is its inability to use existing delineation (RTStructure) (Ganeshan et al., 2008; Davnall et al., 2012).\n\nThe RaCaT is radiomic software that implemented the IBSI defined feature but is not available as a GUI tool as well as unable to perform batch processing. The PyRadGUI software is a GUI-based tool and it can implement batch processing of DICOM images and RT structure for radiomic extraction. As this software is an extension of the pyradiomic package it inherently implements the IBSI feature definition. It can extract radiomic features from hundreds of patientsâ images and RTStructure in batch processing mode and store the result in CSV format. Although we have not compared the delineation converted to NRRD using Plastimatch and 3D Slicer, radiomic feature values comparison show excellent agreement (ICC=0.998±0.012) between the two methods. As our results show, this software calculates radiomics features accurately and reliably. Radiomic extraction from PET and CT images takes a much longer time compared to MRI images, as PET and CT have whole-body images [contains more data] and MRI has regional images [contains less data]. We have used Pyradiomics, open-source software for radiomic extraction in our research infrastructure because this infrastructure can easily be replicated in other research centers. This software works as a plug-in and has no dependencies on the pyradiomic package version, it can be upgraded as and when a new pyradiomic package is available. Customization is the unique feature of this software, which provides flexibility to the user to customize the parameters in the âyamlâ file of the pyradiomics package. The ability of our software to customize and extract 1093 radiomic features from medical images in batch processing enables faster processing of radiomic extraction and storage of feature values in CSV format. During the customization, the user can also select a specific group of features to be extracted. The advantage of this software is its GUI and GUI-based customization of extraction, which allows performing the entire task from the GUI console by clicking the available buttons on the console. The CSV format in which this software stores data where each column represents radiomic features and rows represent individual patientâs data makes it easier to be utilized for machine learning. It can also be concatenated with clinical data if required. Log files can be used for identifying any error that occurs during the processing. We can identify the specific data that contains errors and then take corrective action by referring to the error log of each patient's data that is generated and stored in log file. In our existing project, we have also developed artificial intelligence (AI) infrastructure for AI-based research in oncology and PyRadGUI is also an integral part of it. The PyRadGUI can be implemented as standalone as well as part of AI infrastructure for radiomic based research. The portability and easy installation of this software will encourage the radiomic community to use this software and this software can be a valuable addition to radiomic based research infrastructure.\n\nThere are also a few limitations of this software like it is unable to display the image before or during the procedure. It requires a DICOM image as well as structure for radiomic processing. Future work will be to test this software on Linux operating system, add a statistical and prediction analytics module and image segmentation and display module to this tool.\n\n\nConclusion\n\nWe successfully implemented and validated, PyRadGUI, a GUI-based easy-to-use Radiomic extraction software. This software can easily be implemented on Windows systems. The extracted features using this software are meeting the IBSI standards. We have found this software able to perform batch processing of up to 100 patients and extract radiomic features and store it in ready-to-use CSV format for machine learning. Documentation including the description of how to install and use this software can be found on GitHub (https://github.com/Bionic-TMH/PyRadGUI).",
"appendix": "Data availability\n\nThe corresponding author has access to the data created and used in this study for verification. Data sharing on a public repository is prohibited by IRB permission. Only the project partners are permitted to access the data due to confidentiality reasons regarding patient data.\n\n\nAcknowledgement\n\nThe authors wish to thank The Netherlands Enterprise Agency (RVO) and MeITy for the Indo-Dutch NWO/MeITy BIONIC and TRAIN project.\n\n\nReferences\n\nAerts HJ, Velazquez ER, Leijenaar RT, et al.: Decoding tumour phenotype by noninvasive imaging using a quantitative radiomics approach. Nat. Commun. 2014 Jun 3; 5: 4006. Erratum in: Nat Commun. 2014;5:4644. Cavalho, Sara [corrected to Carvalho, Sara]. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgyeman AA, Ofori-Asenso R: Perspective: Does personalized medicine hold the future for medicine? J. Pharm. Bioallied Sci. 2015 Jul-Sep; 7(3): 239â244. PubMed Abstract | Publisher Full Text | Free Full Text\n\nApte AP, Iyer A, Crispin-Ortuzar M, et al.: Extension of CERR for computational radiomics: a comprehensive MATLAB platform for reproducible radiomics research. Med. Phys. 2018; 45: 3713â3720. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCaers J, Withofs N, Hillengass J, et al.: The role of positron emission tomography-computed tomography and magnetic resonance imaging in diagnosis and follow up of multiple myeloma. Haematologica. 2014 Apr; 99(4): 629â637. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavatzikos C, Rathore S, Bakas S, et al.: Cancer imaging phenomics toolkit: quantitative imaging analytics for precision diagnostics and predictive modeling of clinical outcome. J. Med. Imaging. 2018; 5: 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavnall F, Yip CS, Ljungqvist G, et al.: Assessment of tumor heterogeneity: an emerging imaging tool for clinical practice? Insights Imaging. 2012 Dec; 3(6): 573â589. 2012 Oct 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerlay J, Colombet M, Soerjomataram I, et al.: Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int. J. Cancer. 2019 Apr 15; 144(8): 1941â1953. PubMed Abstract | Publisher Full Text\n\nGaneshan B, Miles KA, Young RC, et al.: Three-dimensional selective-scale texture analysis of computed tomography pulmonary angiograms. Investig. Radiol. 2008 Jun; 43(6): 382â394. PubMed Abstract | Publisher Full Text\n\nGoodwin J, Neugent ML, Lee SY, et al.: The distinct metabolic phenotype of lung squamous cell carcinoma defines selective vulnerability to glycolytic inhibition. Nat. Commun. 2017 May 26; 8: 15503. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGotz M, Nolden M, Maier-Hein K: MITK Phenotyping: an open-source toolchain for image-based personalized medicine with radiomics. Radiother. Oncol. 2019; 131: 108â111. PubMed Abstract | Publisher Full Text\n\nGriethuysen JJM, Fedorov A, Parmar C, et al.: Computational Radiomics System to Decode the Radiographic Phenotype. Cancer Res. 2017; 77(21): e104âe107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaider SP, Burtness B, Yarbrough WG, et al.: Applications of radiomics in precision diagnosis, prognostication and treatment planning of head and neck squamous cell carcinomas. Cancers Head Neck. 2020; 5: 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaralick RM, Shanmugam K, Dinstein I: Textural Features for Image Classification. IEEE Trans. Syst. Man Cybern. 1973; SMC-3: 610â621. Publisher Full Text\n\nJohnson MCI: The ITK Software Guide: Introduction and Development Guidelines. 2015.2015.\n\nLambin P, Rios-Velazquez E, Leijenaar R, et al.: Radiomics: extracting more information from medical images using advanced feature analysis. Eur. J. Cancer. 2012 Mar; 48(4): 441â446. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLimkin EJ, Sun R, Dercle L, et al.: Promises and challenges for the implementation of computational medical imaging (radiomics) in oncology. Ann. Oncol. 2017; 28: 1191â1206. PubMed Abstract | Publisher Full Text\n\nNioche C, Orlhac F, Boughdad S, et al.: LIFEx: A Freeware for Radiomic Feature Calculation in Multimodality Imaging to Accelerate Advances in the Characterization of Tumor Heterogeneity. Cancer Res. 2018; 78: 4786â4789. PubMed Abstract | Publisher Full Text\n\nPfaehler E, Zwanenburg A, de Jong JR , et al.: RaCaT: An open source and easy to use radiomics calculator tool. PLoS One. 2019 Feb 20; 14(2): e0212223. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShi Z, Traverso A, van Soest J , et al.: Technical Note: Ontology-guided radiomics analysis workflow (O-RAW). Med. Phys. 2019 Dec; 46(12): 5677â5684. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStewart CL, Warner S, Ito K, et al.: Cytoreduction for colorectal metastases: liver, lung, peritoneum, lymph nodes, bone, brain. When does it palliate, prolong survival, and potentially cure? Curr. Probl. Surg. 2018 Sep; 55(9): 330â379. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzczypinski PM, Strzelecki M, Materka A, et al.: MaZdaâa software package for image texture analysis. Comput. Methods Prog. Biomed. 2009; 94: 66â76. PubMed Abstract | Publisher Full Text\n\nYip SS, Liu Y, Parmar C, et al.: Associations between radiologist-defined semantic and automatically computed radiomic features in non-small cell lung cancer. Sci. Rep. June 2017; 7 (1): 3519. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang L, Fried DV, Fave XJ, et al.: IBEX: an open infrastructure software platform to facilitate collaborative work in radiomics. Med. Phys. 2015; 42: 1341â1353. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZwanenburg A: EP-1677: Multicentre initiative for standardisation of image biomarkers. Radiother. Oncol. 2017; 123: S914âS915. Publisher Full Text\n\nZwanenburg A, Leger S, ValliÚres M, et al.: Initiative for the IBS. Image biomarker standardisation initiative.2016; 295: 328â338. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "211376",
"date": "31 Oct 2023",
"name": "Nguyen Quoc Khanh Le",
"expertise": [
"Reviewer Expertise Radiomics",
"artificial intelligence"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. The authors compared radiomic feature values between different software tools. However, they lacked specific details on the metrics used for this comparison. The readers would benefit from knowing the statistical methods employed and the criteria for determining \"excellent agreement\" (e.g., ICC=0.998±0.012).\n2. The software limitations mentioned, such as the inability to display images before or during the procedure, may be crucial for users. Elaborating on these limitations and their potential impact on the usability and applicability of the software is necessary.\n3. The use of a DICOM image as well as structure for radiomic processing raises concerns about the software's flexibility. A discussion on the potential limitations imposed by this requirement and the implications for users dealing with non-DICOM data should be included.\n4. The intention to test the software on a Linux operating system is mentioned as future work. It would be helpful to provide insights into the challenges and considerations involved in adapting the software to different operating systems, as this can significantly impact its accessibility.\n5. The authors should provide more details on the specific functionalities and capabilities of statistical and prediction analytics module.\n6. Similarly, the mention of adding an image segmentation and display module is a significant enhancement. The authors should discuss the rationale behind this addition and its potential impact on the software's utility.\n7. While the software is mentioned as a valuable addition to radiomic-based research infrastructure and as part of an AI infrastructure, more details on how PyRadGUI integrates with AI models, its compatibility with different machine learning frameworks, and examples of successful integration with AI-based projects would strengthen the study.\n8. The study could benefit from insights into the user experience, including user feedback, challenges faced during implementation, and any user training required.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "211383",
"date": "31 Oct 2023",
"name": "Seyyed Ali Hosseini",
"expertise": [
"Reviewer Expertise Machine/Deep Learning Quantitative Imaging Radio/Genomics Neuroscience Radiomics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall Comments:\nThe manuscript presents \"PyRadGUI,\" a GUI-based software tool designed to facilitate radiomic feature extraction from medical images. The software aims to address the challenges posed by existing radiomics tools, particularly the lack of user-friendliness and standardization. The authors provide a detailed description of the software's development, features, and validation, along with potential use cases. The manuscript is well-structured, and the software appears promising. However, there are some areas that require further clarification and improvement.\nGeneral Comments:\nClarity and Organization: The manuscript is generally well-organized and clear. However, some sections could benefit from further clarification. For instance, the introduction is lengthy, and while it provides useful background information, it could be more concise. It's essential to maintain a balance between providing context and keeping the reader engaged.\nAbstract: The abstract effectively summarizes the key points of the manuscript. However, it would be helpful to include specific results or findings from the study, as this would give readers a quick overview of the software's performance.\nKeywords: The chosen keywords are appropriate and relevant to the topic.\nEthical Considerations: The authors mention ethical approval for their study but do not provide any information regarding patient consent or data privacy. It would be beneficial to clarify how patient data were handled, especially regarding privacy and consent, as this is an essential ethical aspect of medical research.\nSoftware Availability: The manuscript mentions the availability of the software on GitHub and provides a DOI link. However, it would be helpful to include a brief section in the manuscript explaining how readers can access and use the software.\nSpecific Comments:\nIntroduction: The introduction provides a comprehensive overview of radiomics and its significance. However, it could be condensed to maintain reader engagement. Also, it would be useful to include a clear statement of the study's objectives and the problem the software aims to address.\nMethods: The methods section is detailed and provides a good understanding of how the software was developed and tested. However, there are several grammatical errors and awkward sentences throughout this section that need editing. For example, \"Although we have not compared the delineation converted to NRRD using Plastimatch and 3D Slicer, radiomic feature values comparison show excellent agreement (ICC=0.998±0.012) between the two methods.\" This sentence needs clarification.\nResults: The results section provides valuable information regarding software validation and performance testing. However, it could be enhanced by presenting the results in a more organized and visually appealing manner, such as tables or figures.\nDiscussion: The discussion section effectively highlights the advantages of PyRadGUI and its potential applications. It would be beneficial to address the limitations and future improvements in more detail. For instance, the authors mention future work to add statistical and prediction analytics modules and image segmentation and display modules, but elaborating on these points would provide more insight.\nConclusion: The conclusion appropriately summarizes the key findings and contributions of the study. However, it would be helpful to reiterate the software's main advantages briefly.\nFigures and Tables: Figures and tables are crucial for presenting results and data. Consider adding more visual elements to help readers grasp the key points more easily.\nLanguage and Grammar: The manuscript contains numerous grammatical errors and awkward sentences. Careful editing and proofreading are necessary to improve readability.\nSoftware Citation: Encourage authors to cite the software appropriately in their publications, as this is important for acknowledging the work of software developers.\nSoftware Accessibility: Provide clear instructions on how to access and use the software. This should include information on system requirements, installation, and a brief tutorial if possible.\nIn summary, the manuscript presents a promising software tool for radiomics, but it requires editing for clarity, grammar, and organization. Additionally, providing more information on ethical considerations and access to the software would enhance the manuscript's completeness.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "219099",
"date": "22 Nov 2023",
"name": "Khashayar Namdar",
"expertise": [
"Reviewer Expertise Artificial Intelligence",
"Brain Cancer",
"Statistics",
"Neuroscience",
"MRI"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper has been already reviewed and the current version is in an acceptable stage in terms of writing and structure. As a reviewer, I do not intend to add unnecessary comments. Anything I highlight is for the sake of improving the paper and helping the authors and thus it is open for discussion. Also, the authors are not expected to follow the inefficient tradition of starting every sentence with âwe thank the reviewer for their helpful commentâ.\nFundamental comments:\nThe core of the software is PyRadiomics. As it is mentioned in the manuscript, PyRadiomics is a reliable and popular library for radiomics extraction. Hence, choosing PyRadiomics for the back-end of the software is wise. However, the assumption of âPyRadiomics is overcomplicated therefore researchers will use our toolâ may be superficial. A) this is not a new problem. Cisco routers have been known for their complex terminals and multiple GUI-enabled solutions have been proposed for them. However, still the terminal is the most popular interaction means. Therefore, you need to select your target users and develop the tool for them. B) with LLMs writing advanced scripts, radiomics extraction using PyRadiomics becomes more and more accessible. This is why I do not expect your paper will have many citations. C) As it is mentioned in the manuscript, there are multiple alternatives. The question is not ICC of the extracted features. Those check points are needed, but they are nothing more than sanity checks. The question is âwhy PyRadGUI and not Slicer+PyRadiomics extension?â You answered this question partially by highlighting batch processing. You would need more features D) installation of the libraries is always an issue, especially for researchers with limited technical background (which are your target users). Dockerizing the software and selecting a web interface could be a more versatile approach.\nRadiomics-based ML pipelines are more comprehensive than radiomics extraction. Authors have tried to include more in the software by adding data-loading. However, this makes the solution project-specific. They should decouple feature extraction so that the software becomes compatible with different settings. What if the starting point is not DICOM files? What if there are no RT Structures? What if NifTi, NRRD, or even 2D JPEG/PNGs are provided?\nTechnical Considerations: The total number of extracted features can be more than 1093. You may want to read âOpen-radiomics: A Collection of Standardized Datasets and a Technical Protocol for Reproducible Radiomics Machine Learning Pipelinesâ and check https://openradiomics.org/. Please make sure trimesh is installed.\nThe quality of the figures is low. It seems in Figure 2, Bin Width is misspelled.\nRe. âTo start PyRadGUI, GUI_batch_radiomics.py is run on the command prompt.â: A) An executable file could be provided to make the solution more âsoftwareâ than a tkinter python script. B) the specific file name is introduced out of the context and when the reader has no knowledge of the script structures. Please start the paragraph with introducing the github repository and the scripts and directory structures and functions.\nPyRadiomics has numerous settings and hyperparameters, and you have provided access to several common ones. A) mention what they are why you have decided to choose them B) It would be nice to incorporate an âadvancedâ tab for users with more experience\nWriting: I would improve the paper is the use of words such as âmanyâ and âseveralâ was more controlled. It becomes more important when these words are repeated in the same sentence (e.g. â Several open-source and licensed software packages for radiomic extraction, like IBEX, RaCaT, CaPTK, LifeX or CGITA, Pyradiomics, and TexRad have been developed and used by several researchers in the pastâ).\nAlso, choosing âlikeâ as a replacement for âsuch asâ is informal in academic writing.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-259
|
https://f1000research.com/articles/12-257/v1
|
09 Mar 23
|
{
"type": "Research Article",
"title": "Association between mutant E6 and E7 Human Papilloma Virus Type 16 oncogenes with cyclin D1 expression in cervical cancer: Observational study in Bali",
"authors": [
"I Nyoman Bayu Mahendra",
"I Nyoman Gede Budiana",
"I Gede Mega Putra",
"Anom Suardika",
"Anak Agung Gede Putra Wiradnyana",
"Alisza Novrita Sari",
"I Nyoman Gede Budiana",
"I Gede Mega Putra",
"Anom Suardika",
"Anak Agung Gede Putra Wiradnyana",
"Alisza Novrita Sari"
],
"abstract": "Background: Incidence of cervical cancer is higher in low- and middle-income countries, including Indonesia. In Asia, Indonesia ranks second both in mortality and incidence of cervical cancer. Infection by Human Papilloma Virus (HPV) type 16 is a known risk factor, especially with mutations of the E6 or E7 oncogene that lead to chronic infection and malignancy. Increase in cyclin D1 expression is found on cervical cancer cells, including those resistant to chemotherapy. The role of cyclic D1 in increasing cellular proliferation and decreasing apoptosis leads to cervical cancer. Methods: This study was a cross-sectional observational study carried out between August 2020 until August 2021 at Prof. Dr. I.G.N.G. Ngoerah Hospital, Denpasar, Bali, Indonesia. Samples were collected by consecutive sampling of eligible patients. Polyclinic patients newly diagnosed with cervical cancer and who gave informed consent were further examined. We determined their oncogene E6 and E7 HPV type 16 mutation patterns and their association with cyclin D1 expression using contingency coefficient correlation test. Other outcomes including age distribution, parturition history, BMI, pathological type, clinical stage, and E6/E7 mutation characteristics was also recorded. Results: We included 31 eligible subjects with cervical cancer and HPV type 16-positive, divided into mutant E6/E7 (n=12/31; 38.7%) and wild type (n=19/31;61.3%). Primary outcome of association between mutant oncogene and cyclin D1 expression was weak (c=0.283; p-value=0.1). Secondary outcome showed majority of age on 50 years old or over; normal or overweight BMI; squamous cell carcinoma was the most commonly found (n=28/31; 90%); and clinical stage II (n=18/31; 38%) in both groups. Conclusions: This study concluded that there is no association between mutation in the E6/E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer.",
"keywords": [
"cervical cancer",
"oncogene",
"cyclin expression",
"HPV type 16",
"observational study",
"gynecological cancer"
],
"content": "Introduction\n\nThe estimated incidence of cervical cancer in the world is 13.1 every 100,000 women. The incidence of cervical cancer is found to be higher in lower- and middle-income countries like Indonesia. In 2018, Indonesia ranked second in the incidence and mortality rate of cervical cancer in Asia1,2\n\nOne of the known risk factors of cervical cancer is the Human Papilloma Virus (HPV). HPV types 16 and 18 are the most common types found in cases of cervical squamous cell carcinoma. The HPV oncoproteins that play a role in the development of cervical cancer are E6 and E7.3â5\n\nE6 and E7 initiate a series of processes leading to chronic infection and transformation into cervical cancer. E6 causes degradation of p53, and p21 inhibition leads to a decrease in cellular apoptosis. E7 inactivates pRb so that infected cells experience increased cell proliferation, causes an aberrant synthesis phase of the cell cycle, and increases transcription factor E2F. The sequences carried by E6 and E7 lead to an increase in cyclin D1 expression.5â7\n\nIncrease in cyclin D1 expression is found in cervical cancer cells, including those resistant to chemotherapy.8â10 Cyclin D1 overexpression can augment cell cycle and increase cellular proliferation. The expression of Cyclin D1 also affects the function of apoptosis, hypertrophy and differentiation. Cyclin D1 is thought to have mitogenic activity so as to stimulate hypertrophic growth.11\n\nDetermination of E6 and E7 mutations for HPV type 16 can be useful in the development of HPV molecular diagnostics, prevention, and therapeutic efforts to control or eliminate cervical cancer. Moreover, the development of agents to target cyclin D1 activity, directly or indirectly, by targeting cyclin-dependent kinase (CDK) has also been studied. These agents are used concurrently with chemotherapy and have been shown to reduce resistance to chemotherapy and increase patient survival rates12â14 when patients are referred for chemotherapy or radiotherapy.15,16 Given their significance the association between cylin D1 expression and the E6 and E7 oncogene mutation of HPV type 16 infection as cervical cancer risk factor, they are crucial in understanding cervical cancer. Thus, this research was carried out.\n\n\nMethods\n\nAn observational analytic, cross-sectional study was conducted from August 2020 until August 2021 at the Gynecology polyclinic of RSUP Prof. Dr I.G.N.G. Ngoerah, Denpasar laboratory of microbiology, faculty of medicine Universitas Udayana (FK UNUD), Denpasar and laboratory of histology FK UNUD, Denpasar. This study was approved by the Ethical Board for Research of FK UNUD, Denpasar on 21 December 2021 with protocol number 2021.02.1.1247.\n\nThe sample size was calculated with the following formula:\n\nN=Minimum sample size\n\nZα=Confidence interval with α score of 5% and Zα is 1.960\n\nP=Proportion of oncogene E6 and E7 mutation of HPV 16 was around 70% based on a study by Zhe et al. (2019)17\n\nQ=1-p (proportion of oncogene E6 and E7 of HPV 16 which have compability with the prototype), around 30%\n\nd=Error limitation or absolute precision, 18%\n\nData was collected from cervical cancer patients who visited the polyclinic between August 2020 until August 2021. The sampling method used was consecutive sampling of the population and included patients who fulfilled the eligibility criteria. Patient eligibility was assessed with inclusion and exclusion criteria and provided written informed consent as seen in Table 1. Patient eligibility was assessed with inclusion criteria such as first-time cervical cancer diagnosis during the study setting; not having received surgery, chemotherapy, or radiotherapy. The exclusion criteria were cervical cancer that was not caused by HPV type-16 and if the cervical cancer tissue samples were damaged or not representative for DNA PCR or immunohistochemistry examination. Eligible patients were asked for and provided written informed consent.\n\nEligible patients were asked for demographic characteristics, clinical history, received physical examination, and pathological examination of cervical cancer cells. Patient characteristics such as age, parturition history, body mass index (BMI), cervical cancer clinical stage, and pathological findings were recorded. Cervical cancer tissue specimens were tested for oncogene E6 or E7 mutation by HPV DNA PCR The PCR analyses were conducted at our institution laboratory, DNA isolation was carried out with Roche DNA extraction kit following the product standard protocol. After extraction, a PCR was performed to determine positive and negative HPV with primers My09 (5â²-CGT CCM ARR GGA WAC TGA TC-3â²) and My11 (5â²-GCM CAG GGW CAT AAY AAT GG-3â²). Meanwhile, DNA PCR for HR-HPV type-16 were performed with primers (Forward: 5â²-GAC CCA GAA AGT TAC CAC AG-3â² and reverse: 5â²-CAC AAC GGT TTG TTG TAT TG-3â²). The HPV HR Type 16 PCR program is the same as the PCR program for universal HPV. To determine the quality of the extracted DNA, a PCR was performed for the betaglobin gene with the primers (Forward 5-CAA CTT CAT CCA CGT TCA CC-3 and reverse: 5-GAA GAG CCA AGG ACA GGT AC-3); The PCR results were then compared with the wild type gene on GenBank according to sequence K02718/HPV16R. Amplification of E6 and E7 gene was done with primer sequences as follow: upstream, 5â²AAG GGC GTA ACC GAA AT3â²; downstream, 5â²TCC ATT ACA TCC CGT ACC CTC3â² (Primer 2 OD; 1 OD=33 ÎŒg). The primer was dissolved up to 100 mmol/L at a concentration of 10 ÎŒM (M=mol/L). The PCR reaction mixture of E6 and E7 genes (25 ÎŒL) wasas follows: 1 ÎŒL DNA template (1:20), 1 ÎŒL upstream primer (10 ÎŒM), 1 ÎŒL downstream primer (10 ÎŒM), 12.5 ÎŒL of 2à Taq PCR MasterMix, and double distilled water. The PCR cycle conditions were as follows: pre-denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds; and final extension at 72°C for 5 minutes. After PCR was completed, 5 mL of sample were analyzed by electrophoresis using 1.5% agarose gel; The MEGA6 software was utilized for the sequencing of the E6 and E7 genes. BLAST software was used to compare the nucleotide sequences of the E6 and E7 genes to the HPV 16 prototype (HPv16.P, GenBank access code: NC 001526) from the European variation; cyclin D1 expression was measured by immunohistochemistry with rabbit primary ERβ polyclonal antibodies (BY-02101, Yueyan Biotechnology, Shanghai, China) and rabbit secondary polyclonal antibodies tagged with horseradish peroxidase (K500711, Gene Biotechnology, Shanghai, China) and categorized by Immunoreactive Scoring System (IRS); diagnosis of cervical cancer was made based on histopathological findings with histological classification such as squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma.\n\nAfter the patient was diagnosed with cervical cancer, they were grouped according to E6 or E7 oncogene mutation and each group was tested for cyclin D1 expression. The data was analyzed with IBM SPSS for Windows version 22.0. The correlation between oncogene E6/E7 mutation and cyclic D1 expression was analyzed with a contingency coefficient correlation test. The result ranges from 0 until 1, 0 indicating no correlation and 1 a very strong correlation.\n\n\nResults\n\nFrom August 2020 until August 2021, a total of 100 cervical cancer patients was tested for HPV 16. 31 samples tested positive for HPV 16 with oncogene E6/E7 mutation distribution of wild type of n=19; 61%, and mutated oncogene n=12; 39%. Table 2 summarizes the demographics and characteristics of sample including age, parturition history, BMI, cervical cancer histological classification, and cancer stage which were distributed normally. Mean age for wildtype and mutant type were 50 and 54.5 years old, respectively. Mean body mass index for wildtype was 23.7 and 22.1 for the mutant. Squamous cell carcinoma was the most common histological findings, and cancer clinical stage II was the most common in both types.\n\nTable 3 summarize the characteristic and proportion of E6 and E7 mutation on this study. The proportion of E6 mutants in this study group was 25.8% (8/31), while in the E7 group it was 12.9% (4/31). The population characteristics of the E6 mutation group were dominated by the nucleotide position of 27 prototype T variant C (n=5; 16.1%) followed by position 360 prototype A variant G (n=2; 6.4%) and position 371 prototype G variant A (n=1; 3.2%). The mutation in strain E6 is a synonymous mutation and G371A/R124K the only non-synonymous mutation. In the E7 group, the same proportion was obtained, namely one sample (3.2%) for each nucleotide position, prototype and variant. At E7 the majority of mutations were non-synonymous 9.6% (3/31) in N29T; N29S; R77C.\n\nThe association of mutations in the E6 and E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer is summarized in Table 4. There was no association between mutations in the E6/E7 HPV type 16 oncogene with cyclin D1 expression (c=0.283; p value=0.1). The correlation coefficient between the mutations of the E6 and E7 HPV type 16 oncogenes with Cyclin D1 expression was weak, as seen in Table 4.\n\n\nDiscussion\n\nIn this study, we described the association of mutations in the E6 and E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer. Our study included 31 patients with cervical cancer and HPV type 16 infection based on pathological examination and HPV DNA PCR. The samples median age was 54.5 (21-58) years old, in accordance with a study in Denmark and Sweden with a higher incidence of cervical cancer in patients aged 50 years or older.18,19 Women aged 50 years and over are usually in the perimenopausal or postmenopausal phase; therefore, their physiological and pathological characteristics differ significantly from women of childbearing age.20\n\nThe mean BMI was 22.1 kg/m2 in cervical cancer patients with mutant oncogenes E6 and E7 with a BMI range of 19.5-28.2 kg/m2. In this study, patients were found to be in the ideal BMI and overweight categories. The effect of body mass index (BMI) on precancerous cervical lesions (PCL) and cervical cancer is not clear. There are claims that underweight women may have an increased risk of cervical lesions while overweight women have a reduced risk of PCL compared to women with a normal BMI.21 One of the identified risk factors for PCL is being underweight (BMI<18.5 kg/m2), so the probability of being positive for PCL was fourfold higher among underweight women than women with a normal BMI. This relationship can be explained by the possible association between low BMI, poor nutritional status and low immunity. Therefore, participants with a low BMI tend to be malnourished, which in turn can compromise their immunity.22\n\nThe HPV genome consists of small, conserved double-stranded DNA with an approximate size of 8000 base pairs, and consists of three regions. The molecular biology of this tiny DNA molecule is complex. There are six starting proteins, three regulatory proteins (E1, E2, and E4) and three oncoproteins (E5, E6, and E7) encoded in 4000 base pairs that participate in viral replication and cell transformation.23\n\nIn HPV16-positive cells, it was found that viral genes E6 and E7 remained integrated into the host genome and expressed, although in some HPV16-infected cells, E6/E7 overexpression could be absent. In addition, E6/E7 overexpression was also found in cells infected with other HPV types. E6 and E7 are small proteins composed of 100 to 150 amino acids with no known enzymatic activity; they can affect host cell activity when they bind to cell receptor proteins. E6, for example, binds to E6-associated protein (E6AP), a ubiquitin ligase that causes structural changes in E6 that allow it to bind to p53, a tumor-inhibiting protein in a cell cycle control manner to form the E6/E6AP/p53 complex. Therefore, E6 and E7 are important factors in the occurrence of cervical cancer in HPV16-positive cells.24\n\nThe HPV genome may integrate with the host genome or remain in an episomal form, with 83% of cases of HPV-positive cervical cancer showing evidence of integration of the HPV genome into host cells. In the case of integration of the viral genome with the host genome, it often leads to disruption of the E2 gene site. The E2 gene is responsible for suppressing E6 and E7, thereby causing E6 and E7 to be activated upon integration of the viral genome into the host genome. Throughout the course of infection, the activities of E6 and E7 are responsible for the doubling of the viral genome with the help of cellular machinery. They can trick cells into becoming oncogenic in the process of viral replication. Therefore, HPV-mediated tumor development can be defined as collateral damage from viral infection.25\n\nThe E6 protein is thought to increase cell proliferation by stimulating the degradation of the tumor suppressor protein p53 through the formation of a complex consisting of E6, p53 and the cellular ubiquitination enzyme E6-AP. E6-stimulated degradation interferes with the biological function of p53, thereby interfering with the control of cell cycle progression, which ultimately leads to increased tumor cell growth. Although it is generally accepted that the ability of high-risk HPV types E6 to target p53 for degradation contributes to virus-induced cellular transformation, it is also clear that the E6 protein has oncogenic activity independent of p53.26\n\nThe E7 protein encoded by high-risk HPV types, such as HPV 16 and HPV 18, binds to the Rb protein with much higher affinity than that encoded by low-risk HPV types, such as HPV type 6 and HPV type 11. E7 binds to the Rb protein with the region being called âpocket domainâ. The âpocket domainâ sequence of Rb is critical for its tumor suppressor function, with many naturally occurring Rb loss-of-function mutations appearing clustered within this âpocket domainâ. One of the major biochemical functions of Rb is to bind to transcription factors of the E2F family and suppress the gene expression of the replication enzymes. As a result of mutations in E7 resulting in loss of function of Rb resulting in loss of replication suppression so that cells continue to replicate and cause cancer.27\n\nTo ensure continued cell proliferation, HPV-infected cells also need to pass the mitotic test, which is also carried out by E6 and E7 simultaneously. E6 is dependent on a p53-dependent pathway, while E7 avoids the spindle checkpoint in a p53-independent manner, with the help of pRb. As a result of alterations in several cell cycle regulators, the cyclin-CDK complex is a major player of a dramatically altered cell cycle in HPV-infected cervical cancer cells. Cyclin D1-CDK4 and cyclin D1-CDK2 associations is reduced in E6-expressing cells and completely abolished in E6 and E7-expressing cells.28\n\nThere was not statistically significant difference between the mutant and wild-type groups. This indicates that the mutant and wild-type have a weak correlation with the emergence of cyclin D1 expression and mutation or lack thereof does not determine whether the mutant or wild-type has a higher or less cyclin D1 value. Cancer development and progression involve activation of oncogenes, inactivation of tumor suppressor genes, and imbalance of immune system regulatory mechanisms, a combination which causes cellular variability and leads to tissue transformation into cancer. Cyclin, and especially cyclin D1, functions as a cell cycle regulator and promotescell proliferation. Mutation or over expression of Cyclin D1 may alter the cell cycle in various cancers.27\n\nMany studies have focused on the expression of cyclin D1 and its junction variants cyclin D1a and cyclin D1b, which arise from single nucleotide polymorphisms (SNPs) in the CCND1 gene in cervical cancer. While cyclin D1 has been associated with different clinical and pathological stages of cervical cancer, few studies have focused on its correlation with cervical cancer prognosis. Currently, the accepted view is that Cyclin D1 and its isoforms play an important role in the development and progression of cervical cancer. During the normal cell cycle, cyclin D1 forms a complex with CDK4, which promotes phosphorylation of the tumor suppressor protein. The transcription factor E2F is then initiated to promote DNA synthesis, allowing the completion of cell division by moving from the G1 phase to the S phase. cyclin D1 can thus be seen as a shortening of the G1 phase of the cell cycle. When the control of the cyclin D1 protein is abnormal and several cancer-associated genes result in an increase in its expression, the time the cell spends in the G1 phase of the cell cycle is significantly reduced, causing the cell to enter the S phase earlier, which in turn results in uncontrolled cell proliferation and transformation leading to carcinogenesis. Currently, cyclin D1 is recognized as a proto-oncogene, and its overexpression can alter progression through the cell cycle, leading to uncontrolled cell proliferation and malignancy. Unlike the findings of this study, in a study to detect Cyclin D1 expression, it was found that there was a fourfold increase in Cyclin D1 expression in cancer conditions. But in this study, the comparison of the cancer group was normal cells (no malignancy activity at all) and the research focused on uterine cancer.28\n\nResearch by Park et al. (2016) supports the result of our study. Their study stated that there is a role for glycogen synthase kinase 3β (GSK3β), a pluripotent protein kinase that is involved in cancer development through the regulation of various oncogenic molecules. Cyclin D1, an important regulator of G1 to S phase transition in various cells, is one of the target proteins that GSK3β regulates. Squamous cell carcinoma has an increased expression of GSK3β which plays a role in cervical carcinogenesis and has an inverse correlation with cyclin D1 expression in this process.29\n\nAnother study by Bae et al. (2001) found that decreased cyclin D1 is regulated at the transcriptional level in cervical cancer. Cyclin D1 is less expressed in cervical neoplasia, and is more frequently expressed in malignant lesions. This may be because cyclin D1 is no longer required for G1 progression in HPV-transformed cells due to the binding of HPV E7 to Rb leading to the release of the transcription factor E2F.30 Pyeon et al. (2007) found that there was no significant difference between the increase in cyclin D1 and cervical cancer. In HPV-positive cancers, p16 is expressed at high levels and cyclin D1 at low levels, in contrast to HPV-negative cancers.31 In this study, there was also no relationship between cyclin D1 and the occurrence of mutations in HPV-positive cancer, and cyclin D1 levels were not associated with changes in lesions from pre-cancerous to cancerous.\n\nThe limitation of this study was a limited number of samples we found in a one-year, single centre study. Further study with a greater sample population can provide more accurate result such as on national level.\n\n\nConclusions\n\nThis study concluded that there is no association of mutation in the E6/E7 HPV type 16 oncogenes with cyclin D1 expression in patients with cervical cancer.",
"appendix": "Data availability\n\nFigshare: Data of Cervical Cancer HPV 16 oncogen E6 or E7 with Cyclin D1 expression.xlsx, https://doi.org/10.6084/m9.figshare.21786644.v1. 32\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nBray F, Ferlay J, Soerjomataram I, et al.: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018; 68(6): 394â424. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: Indonesia Source GLOBOCAN 2018. International Agency for Research on Cancer. 2019; 256: 1â2.\n\nWipperman J, Neil T, dan Williams T: Cervical Cancer: Evaluation and Management. Am. Fam. Physician. 2018; 15(7): 43â47. Publisher Full Text\n\nTadlaoui KA, Hassou N, Bennani B, et al.: Emergence of oncogenic high-risk human papillomavirus types and cervical cancer, Emerging and Reemerging Viral Pathogens: Volume 1: Fundamental and Basic Virology Aspects of Human, Animal and Plant Pathogens. Elsevier Inc.; 2019. Publisher Full Text\n\nVonsky M, Shabaeva M, Runov A, et al.: Carcinogenesis Associated with Human Papillomavirus Infection. Mechanisms and Potential for Immunotherapy. Biochem. Mosc. 2019; 84(7): 782â799. PubMed Abstract | Publisher Full Text\n\nDoorbar J: Molecular biology of human papillomavirus infection and cervical cancer. Clin. Sci. 2006; 110(5): 525â541. Publisher Full Text\n\nHoppe-Seyler K, Bossler F, Braun JA, et al.: The HPV E6/E7 Oncogenes: Key Factors for Viral Carcinogenesis and Therapeutic Targets. Trends Microbiol. 2018; 26(2): 158â168. Elsevier Ltd. PubMed Abstract | Publisher Full Text\n\nVan De Putte G, Kristensen GB, Lie AK, et al.: Cyclins and proliferation markers in early squamous cervical carcinoma. Gynecol. Oncol. 2004; 92(1): 40â46. PubMed Abstract | Publisher Full Text\n\nBahnassy AA, Zekri A-R, El-din HA, et al.: The role of cyclins and cyclins inhibitors in the multistep process of HPV-associated cervical carcinoma. J. Egypt. Natl. Canc. Inst. 2006; 18(4): 292â302. PubMed Abstract\n\nLeal-Esteban LC, dan Fajas L: Cell cycle regulators in cancer cell metabolism. Biochim. Biophys. Acta Mol. Basis Dis. 2020; 1866(5): 165715. Elsevier B. V. Publisher Full Text\n\nHaybar H, Shahrouzian M, Gatavizadeh Z, et al.: Cyclin D1: A Golden Gene in Cancer, Cardiotoxicity, and Cardioprotection. Jundishapur J. Chronic Dis. Care. 2021; 10(3): e112413. Publisher Full Text\n\nWhittaker SR, Mallinger A, Workman P, et al.: Inhibitors of cyclin-dependent kinases as cancer therapeutics. Pharmacol. Ther. 2017; 173: pp. 83â105. Elsevier Inc. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlmeida AM, Queiroz JA, Sousa F, et al.: Cervical cancer and HPV infection: ongoing therapeutic research to counteract the action of E6 and E7 oncoproteins. Drug Discov. Today. 2019; 24(10): 2044â2057. PubMed Abstract | Publisher Full Text\n\nRoskoski R: Cyclin-dependent protein serine/threonine kinase inhibitors as anticancer drugs. Pharmacol. Res. 2019; 139: pp. 471â488. Elsevier Ltd. Publisher Full Text\n\nWinata IGS, Hidayat YM, Winarno GN, et al.: Pulsatility Index and Hypoxia Inducible Factor-1α Expression Predict the Clinical Response after External Radiation in Patients with Stage IIB to IVA Cervical Cancer. Asian Pac. J. Cancer Prev. 2019; 20(7): 2073â2078. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWinata IGS, Budiana ING, Jawi IM, et al.: Neoadjuvant Chemotherapy in Stadium IB3, IIA2 and IIB Cervical Cancer a Narrative Review. Biomed. Pharmacol. J. 2022; 15(2): 901â910. Publisher Full Text\n\nZhe X, Xin H, Pan Z, et al.: Genetic variations in E6, E7 and the long control region of human papillomavirus type 16 among patients with cervical lesions in Xinjiang, China. Cancer Cell Int. 2019; 19(1): 1â11. Publisher Full Text\n\nMoore KN, Java JJ, Slaughter KN, et al.: Is age a prognostic biomarker for survival among women with locally advanced cervical cancer treated with chemoradiation? An NRG Oncology/Gynecologic Oncology Group ancillary data analysis. Gynecol. Oncol. 2016; 143(2): 294â301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLynge E, Lönnberg S, Törnberg S: Cervical cancer incidence in elderly women-biology or screening history? Eur. J. Cancer. 2017; 74: 82â88. Publisher Full Text\n\nYan J, He Y, Wang M, et al.: Prognostic Nomogram for Overall Survival of Patients Aged 50 Years or Older with Cervical Cancer. Int. J. Gen. Med. 2021; 14: 7741â7754. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeng YH: The association between obesity and gynecological cancer. Gynecol. Minim. Invasive Ther. 2015; 4(4): 102â105. Publisher Full Text\n\nTaye BT, Mihret MS, Muche HA: Risk factors of precancerous cervical lesions: The role of womenâs socio-demographic, sexual behavior and body mass index in Amhara region referral hospitals; case-control study. PLoS One. 2021; 16(3 March): e0249218âe0249215. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJing Y, Wang T, Chen Z, et al.: Phylogeny and polymorphism in the long control regions E6, E7, and L1 of HPV Type 56 in women from southwest China. Mol. Med. Rep. 2018; 17(5): 7131â7141. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChan CK, Aimagambetova G, Ukybassova T, et al.: Human Papillomavirus Infection and Cervical Cancer: Epidemiology, Screening, and Vaccination - Review of Current Perspectives. J. Oncol. 2019; 2019: 1â11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPal A, dan Kundu, R.: Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy. Front. Microbiol. 2020; 10(January). PubMed Abstract | Publisher Full Text | Free Full Text\n\nYim E-K, Park J-S: The Role of HPV E6 and E7 Oncoproteins in HPV-associated Cervical Carcinogenesis. Cancer Res. Treat. 2005; 37(6): 319â324. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCasimiro MC, Di Sante G, Di Rocco A, et al.: Cyclin D1 Restrains Oncogene-Induced Autophagy by Regulating the AMPK-LKB1 Signaling Axis. Cancer Res. 2017; 77(13): 3391â3405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTripathi R, Rath G, Jawanjal P, et al.: Cyclin D1 protein affecting global womenâs health by regulating HPV mediated adenocarcinoma of the uterine cervix. Sci. Rep. 2019; 9(1): 1â6. Publisher Full Text\n\nPark H, et al.: Glycogen synthase kinase 3β and cyclin D1 expression in cervical carcinogenesis. Obstet. Gynecol. Sci. 2016; 59(6): 470â478. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBae DS, Cho SB, Kim YJ, et al.: Aberrant expression of cyclin D1 is associated with poor prognosis in early stage cervical cancer of the uterus. Gynecol. Oncol. 2001; 81(3): 341â347. PubMed Abstract | Publisher Full Text\n\nPyeon D, Newton MA, Lambert PF, et al.: Fundamental Differences in Cell Cycle Deregulation in Human Papillomavirus-Positive and Human Papillomavirus-Negative Head/Neck and Cervical Cancers. Cancer Res. 2007; 67(10): 4605â4619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMahendra INB, Budiana ING, Putra IGM, et al.: Data of Cervical Cancer HPV 16 oncogen E6 or E7 with Cyclin D1 expression.xlsx. Dataset. figshare. 2022. Publisher Full Text"
}
|
[
{
"id": "170486",
"date": "22 May 2023",
"name": "Jacyara Macedo",
"expertise": [
"Reviewer Expertise Molecular biology of cancer",
"Molecular epidemiology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work was developed in order to investigate possible association of mutated HPV E6 and E7 oncgenes with cyclin D1 expression in cervical cancer cells.\nIntroduction section should be improved. The importance of E6 and E7 oncogenes and the cyclin D1 expression should have been better explored to support the study.\nReferences for the sentence âThe sequences carried by E6 and E7 lead to an increase in cyclin D1 expression.â were not adequate.\nThe sentence âIncrease in cyclin D1 expression is found in cervical cancer cells, including those resistant to chemotherapy.â is not supported by the references 8 and 10. I could not check the reference 9.\nThe sentence below should be rewritten:\nâGiven their significance the association between cylin D1 expression and the E6 and E7 oncogene mutation of HPV type 16 infection as cervical cancer risk factor, they are crucial in understanding cervical cancer. Thus, this research was carried out.â\nMethodology was not properly described.\nThe sentence âPatient eligibility was assessed with inclusion and exclusion criteria and provided written informed consent as seen in Table 1â could be excluded. âTable 1â should be included at the end of the paragraph.\nSome parts were not presented or were partially presented. For example, there are different Roche DNA extraction kits. No information about the kit used for DNA extraction in this work was given. References of primers used in this work were not mentioned. PCR conditions used in the amplification of different regions of the viral genome were not properly described. Details of the immunohistochemistry and DNA sequencing were not described.\nThe results should be better explored. What are the effects of non-synonymous mutations in the HPV E6 and E7 oncogenes in the protein structure/function?\nIn mutated E6 protein, arginine was replaced by lysine. In mutated E7 proteins, asparagine at the position 29 was replaced by hydroxylated aminoacids, threonine or serine, and arginine at the position 77 was replaced by cysteine.\nDo synonymous mutations in the HPV E6 and E7 genes cause any change in protein expression, conformation or function? Is it correct to group all mutations to assess possible associations of E6/E7 mutations with cyclin D1 expression?\nThese issues were not properly discussed. In fact, the limited number of samples did not allow for a deeper discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "307750",
"date": "13 Aug 2024",
"name": "Eric Siegel",
"expertise": [
"Reviewer Expertise I am a biostatistician with 25 years of expertise practicing statistics."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis cross-sectional study enrolled cervical-cancer patients that were positive for HPV type 16. Its two purposes were (1) to characterize the patients' HPV genomes for whether they carried mutations in either the E6 or E7 oncoprotein, and (2) to test for whether the presence of such mutations are associated with Cyclin D1 expression. They obtained 31 subjects that were positive for HPV16, of which 19 had wildtype E6/E7 and 12 had mutated E6/E7. They classified Cyclin D1 expression as Weak or Strong, and tested for association with E6/E7 mutation status using a 2x2 contingency table with an unidentified statistical test, and used the contingency coefficient (c-score) as the association's effect-size measure. They obtained c-score=0.283 with p-value = 0.1 from the unidentified statistical test. From this result, they concluded that there is no association of mutation in the E6/E7 HPV-16 oncogenes with Cyclin D1 expression in patients with cervical cancer. The authors' conclusion is false for two reasons. One, it is a fallacy to conclude that no association exists simply because the p-value is greater than 0.05. The fact that the authors obtained a c-score greater than 0 means that some level of association did exist, but that the study was underpowered to detect it. Furthermore, the fact that the authors obtained a c-score of 0.283 from Table 4 means that the authors also obtained a phi coefficient of ±0.295 from the same Table 4. Phi coefficients of magnitude 0.1, 0.3, and 0.5 were classified respectively as small, medium, and large by Cohen J (1992) Ref 1. By this classification, phi=±0.295 represents a medium association, and therefore, the authors' c-score=0.283 represents an association that is medium, not weak as the authors claimed. Because a medium association, if it exists, could have relevance to public health, it is dangerous to pretend that a medium association equals no association simply because the p-value was greater than 0.05. Two, the authors have a mistake somewhere in their Table 4. The top row of Table 4 shows only 11 cases with mutant E6 or E7, while the bottom row of Table 4 shows 20 cases with wildtype E6 or E7. These numbers are not consistent with the numbers in Table 2, the numbers in the first paragraph of the Results section, or the numbers in the Results section of the abstract. Correcting Table 4 to be consistent with the rest of the manuscript has a 50% chance of pushing the p-value down below 0.05, but also a 50% chance of pulling it up to 0.15 Two other statistical issues. The first issue is, the authors never say what statistical test procedures they used. It appears that they used t-tests and Fisher's exact tests in Table 2, and used the chi-square test in Table 4, but because they never say, the reader cannot tell for sure. The authors need to say what tests they used and what variables they used them on. The authors probably also ought to stop using the chi-square test in Table 4 because of the warnings about expected cell counts being too small. The second statistical issue is this. The authors correctly calculated a \"minimum\" sample size from the formula that they used (page 3 of manuscript), but they used the wrong formula. The sample-size formula they used is the formula for testing a single sample for whether its observed proportion P-hat of mutant E6/E7 is significantly different from the hypothesized constant P0=70% based on Zhe et al. 2019. Moreover, the formula they used treats \"d=error limitation or absolute precision\" as the ±95% margin of error on P-hat given P0, which is the same as saying that the minimum sample size calculated by their formula is the minimum sample size needed to obtain 50% power (not 80% power) to detect the difference between P-hat and P0. But what the authors actually did in this study was, they split their n=31 cases into two sample, determined the number and proportion of strong Cyclin D1 in each sample, and used the chi-square test to compare the two samples to each other for the difference in their strong Cyclin-D1 proportions. To calculate the minimum sample size for that two-sample comparison of Cyclin-D1 proportions will require a different formula from the one the authors used.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "266381",
"date": "17 Sep 2024",
"name": "Atar Singh Kushwah",
"expertise": [
"Reviewer Expertise PharmacoEpiGenetics",
"Clinical Oncology and Biotherapeutics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMahendra et al, demonstrated the relationship between mutations in the E6 and E7 oncogenes of HPV Type 16 and Cyclin D1 expression in cervical cancer patients. The study included 31 cervical cancer patients, 38.7% of whom had mutations in the E6/E7 oncogenes. Despite the known role of these oncogenes in promoting cancer through disruption of cellular processes like p53 degradation and pRb inactivation, the study found no significant association between these mutations and Cyclin D1 expression. Cyclin D1 is commonly overexpressed in various cancers, but this study suggests that its expression in cervical cancer may not be directly influenced by E6/E7 mutations. The findings indicate that other molecular mechanisms may be involved in Cyclin D1 regulation, warranting further research with larger sample sizes to clarify these pathways. While reviewing the manuscript, I found the following limitations that need to be rectified for further consideration:\nThe statistical tests used in the study are not clearly described. It appears that t-tests, Fisher's exact tests, and chi-square tests were used, but this is not explicitly stated. Furthermore, the chi-square test in Table 4 is questionable due to low cell counts, potentially making it unreliable. The sample size formula used is incorrect for the study's objectives. The formula calculates the sample size for a single sample proportion test, but the study involves comparing two groups, requiring a different calculation method. Table 4 contains inconsistencies with the rest of the manuscript, showing 11 cases with mutant E6/E7 and 20 cases with wild-type E6/E7, which do not match earlier data. Key details about the DNA extraction kit, PCR conditions, and immunohistochemistry procedures are missing, making replication difficult. Some references cited to support claims about Cyclin D1 expression are not appropriate or do not back the statements made. The authors concluded no association between E6/E7 mutations and Cyclin D1 expression based solely on a p-value greater than 0.05. However, the c-score (0.283) suggests a medium correlation, indicating that the study may be underpowered rather than the relationship being non-existent. The potential functional consequences of non-synonymous mutations in the E6/E7 oncogenes are not thoroughly discussed. The effects of synonymous mutations on protein structure and function are also not addressed. The study does not offer significant new findings or insights into the relationship between E6/E7 mutations and Cyclin D1 expression. Previous research has explored Cyclin D1's role in cervical cancer, and the study does not significantly advance the current understanding.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-257
|
https://f1000research.com/articles/12-253/v1
|
09 Mar 23
|
{
"type": "Study Protocol",
"title": "Presepsin and procalcitonin as markers of infectious respiratory diseases in children: a protocol for a scoping review of the literature",
"authors": [
"Giorgio Sodero",
"Francesco Mariani",
"Valentina Pulcinelli",
"Carolina Gentili",
"Piero Valentini",
"Danilo Buonsenso",
"Giorgio Sodero",
"Valentina Pulcinelli",
"Carolina Gentili",
"Piero Valentini",
"Danilo Buonsenso"
],
"abstract": "Background: Presepsin is a new inflammatory marker used in clinical practice for the diagnosis of severe bacterial infections, which might be used in combination or substitution of other classical indexes such as procalcitonin. Presepsin has been studied mainly in specific categories of patients (generally adults, critically ill patients or neutropenic children) to identify severe bacterial infections and to predict the mortality risk, while the role of this marker in discriminating bacterial from viral infections in otherwise healthy children of different age groups is less examined. This scoping review aims to better explore available evidence around the potential role of presepsin in pediatric respiratory infectious diseases, analyzing its ability to distinguish the severity and type of respiratory pathology and comparing it with a classic inflammation index such as procalcitonin. Methods: We started our research in February 2023 in the following bibliographic databases: PubMed, EMBASE, Cochrane and SCOPUS. The main review question will be âWhat is known about the diagnostic role of presepsin and/or procalcitoninin the differential diagnosis of respiratory tract infectionâs severity and etiology?â We will include randomized and non-randomized controlled trials, prospective and retrospective observational studies, performed on children and adolescents (younger than 18 years), hospitalized or not, with a confirmed diagnosis of upper and/or lower respiratory infectious disease. We will include children diagnosed with pneumonia, bronchiolitis, bronchitis, croup, and other types of infectious respiratory diseases. To report our findings, we will follow Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist. Dissemination: The findings of this review will be published in a peer-reviewed journal and presented in national and international conferences.",
"keywords": [
"Presepsin",
"infectious disease",
"bronchiolitis"
],
"content": "Introduction\n\nPresepsin is a new marker of inflammation formed by cleavage of N-terminal of soluble CD14, a member of the Toll-like receptors group; this protein is present in macrophage and other granulocyte cells on their cell membranes, and it is responsible for intracellular transduction of endotoxin signals.1 In the last years, presepsin has been increasingly used in clinical practice as an indicator of presence and severity of serious infections like sepsis, as it is released by immune system cells involved in the response to pathogenic bacteria, although its utility in patientsâ prognosis is not yet fully understood, particularly in children.2\n\nIn pediatrics, it has been particularly studied for the early diagnosis of neonatal sepsis, in combination with other classic inflammation markers such as procalcitonin,3 while the role of presepsin in discriminating bacterial from viral infections in other clinical scenarios is less studied. For example, respiratory diseases still represent a major cause of mortality, morbidity and antibiotic use, and in this context presepsin could be used as a useful discriminator of bacterial pneumonia or severity,4 and it could be also related to the mortality risk of critical patients.\n\nAlthough its use in neonates is well characterized,3 the evidence for the use of this marker in children aged > 6 months is not clear because it is not a test routinely used in clinical practice and it has been studied mainly in critically ill patients or with important comorbidities such as neutropenia,5,6 with excellent results.\n\nThis scoping review aims to analyze the use of presepsin and procalcitonin in pediatric respiratory infectious diseases, analyzing the ability to distinguish the severity and type of respiratory pathology. In addition, we will also focus on comparing presepsin with procalcitonin, a better studied marker of severe bacterial infections in children.\n\n\nReview questions\n\nThe main review question will be âWhat is known about the diagnostic role of presepsin and/or procalcitonin in the differential diagnosis of respiratory tract infectionâs severity and etiology?â\n\nThis review will also assess the following sub-questions:\n\n1. Does the adjunction of presepsin to the use of procalcitonin improves the accuracy in identifying bacterial infectious diseases?\n\n2. What is the role of presepsin and procalcitonin in the subgroup of children with bronchiolitis?\n\n3. What is the role of presepsin and procalcitonin in the subgroup of children with respiratory syncytial virus bronchiolitis?\n\n\nInclusion criteria\n\nThis review will include studies performed on children and adolescents (younger than 18 years) with a confirmed diagnosis of upper and/or lower respiratory infectious disease (clinical, microbiological or radiological diagnosis). We will include children diagnosed with pneumonia, bronchiolitis, bronchitis, croup, and other types of infectious respiratory diseases.\n\nThe main concept of this review will be the use of presepsin and procalcitonin in pediatric respiratory infectious diseases.\n\nConsidering the large spectrum of severity of the disease, we expect to find articles involving patients both hospitalized or not for respiratory infections.\n\nThis review will include both randomized controlled trials and non-randomized controlled trials. All types of observational studies, prospective and retrospective (including case-control, cohort and cross-sectional studies, small case series or single case reports) will be included.\n\n\nMethods\n\nWe started our research in February 2023 in the following bibliographic databases: PubMed, EMBASE, Cochrane and SCOPUS.\n\nThere were no date restrictions. Only articles written in English will be included.\n\nPatients younger than 18 years of age were considered as children or pediatric patients. The search strategy for Pubmed is available as supplementary material of this protocol (Appendix 1 available at https://doi.org/10.6084/m9.figshare.22155131.v1); the terms used for this search were adapted for use with other bibliographic databases.\n\nAfter the search, the studies have been exported to Rayyan. A first screen to exclude duplicates was performed by one author.\n\nTitles and/or abstracts of studies retrieved using the search strategy will be screened independently by two reviewers to identify studies that could be inserted in the review. Full texts of potentially eligible studies will be retrieved and independently assessed for eligibility by two reviewers. Each researcher will be blinded to the decision of the other researcher. Any disagreement between them over the eligibility of studies will be resolved through discussion and, in case of further disagreement, by discussion with a third reviewer.\n\nAll the studies that will not meet the inclusion criteria will be excluded and a table with the reason why those studies were excluded will be inserted in the final manuscript.\n\nThe results of the search will be reported in the PRISMA flow diagram.\n\nTwo review authors will extract data independently, everyone on a different Excel spreadsheet. Each researcher will be blinded to the decision of the other researcher. When the process will be completed, in case of discordance, any disagreement will be identified and resolved through discussion (with a third author if necessary).\n\nAn Excel file will be used to store data. When available, extracted information will include:\n\n1. study general features: title, author, year of publication, type of study, number of patients included in the study, geographical area where the study has been performed\n\n2. participant general features: sample size of each group, nationality, age, socio-economic status, comorbidities\n\n3. clinical manifestations of children included in our review\n\n4. main imaging findings: type of lung involvement at chest X-Ray and/or CT scan\n\n5. microbiological results\n\n6. results of the inflammation indices performed (procalcitonin and presepsin)\n\n7. antibiotic use\n\n8. hospitalization, including pediatric intensive care\n\n9. outcomes (death, survival; survival with or without sequelae; type of sequelae)\n\nTo report our findings, we will follow Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist.\n\nWe will produce a narrative synthesis of the findings from the studies included in the review describing the results we have obtained and providing our opinion on their interpretation. For the narrative synthesis we will prefer articles in which etiological diagnosis was specified.\n\nWe will also use tables and charts to summarize both study characteristics and the most important clinical, diagnostics, treatments and outcomes data.\n\nMore specifically, we will summarize our findings using tables. The first one will include the characteristics of included studies (number of studies, study design, year of publication, characteristics of the study populations, and countries where studies were conducted) and the second one will include participant general features. Then we will provide different tables or figures summarizing main data about clinical presentation, type of respiratory disease analyzed, imaging characteristics, hematochemical results with procalcitonin and presepsin levels, treatments performed, outcomes and possible predictors of outcome.\n\nThe findings of this review will be published in a peer-reviewed journal and presented in national and international conferences.\n\nWe performed the research and screened for duplicates.\n\nThere will not be direct patient and public involvement in this review.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAzim A: Presepsin: A Promising Biomarker for Sepsis. Indian J. Crit. Care Med. 2021 Feb; 25(2): 117â118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVelissaris D, Zareifopoulos N, Karamouzos V, et al.: Presepsin as a Diagnostic and Prognostic Biomarker in Sepsis. Cureus. 2021 May 13; 13(5): e15019. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRuan L, Chen GY, Liu Z, et al.: The combination of procalcitonin and C-reactive protein or presepsin alone improves the accuracy of diagnosis of neonatal sepsis: a meta-analysis and systematic review. Crit. Care. 2018 Nov 21; 22(1): 316. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMemar MY, Baghi HB: Presepsin: A promising biomarker for the detection of bacterial infections. Biomed. Pharmacother. 2019 Mar; 111: 649â656. Epub 2019 Jan 3. PubMed Abstract | Publisher Full Text\n\nEl Gendy FM, El-Mekkawy MS, Saleh NY, et al.: Clinical study of Presepsin and Pentraxin3 in critically ill children. J. Crit. Care. 2018 Oct; 47: 36â40. Epub 2018 Jun 3. PubMed Abstract | Publisher Full Text\n\nÃzdemir ZC, DÃŒzenli-Kar Y, Canik A, et al.: The predictive value of procalcitonin, C-reactive protein, presepsin, and soluble-triggering receptor expressed on myeloid cell levels in bloodstream infections in pediatric patients with febrile neutropenia. Turk. J. Pediatr. 2019; 61(3): 359â367. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "234652",
"date": "16 Feb 2024",
"name": "Noémie Boillat-Blanco",
"expertise": [
"Reviewer Expertise ID"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis protocol presents the background and methodology of a scoping review on prepepsin and procalcitonin as markers of infectious respiratory diseases in children. The subject of the study is interesting and relevant because data on this specific topic is missing and prepepsin could be an interesting marker for risk stratification and antibiotic guidance. However, the methodology of the study should be improved. Abstract: Background: Type of respiratory pathology: be more precise Methods: Diagnostic role of prepepsin and/or procalcitonin: do you mean âprepepsin alone or in combination with procalcitoninâ? We are not interested in the performance of procalcitonin alone as they are already many meta-analyses on this topic. Confirmed diagnosis of upper/lower RTIâŠ.and other types of infectious resp disaeses: be more precise, do you plan to include ENT infections? Pharyngitis, sinusitis, otitis?\nIntroduction: Why did you choose a scoping review methodology rather than a systematic review? Analyze the use of prepepsin and/or procalcitonin: be more precise: alone or in combination withâŠ. Type of resp pathology:: what do you mean: differentiate bacterial vs viral RTI?\nReview questions: Prepepsin alone or in combinationâŠ.: please replace throughout the manuscript. In the DD of RTI: lower RTI?\nInclusion criteria: Please, be more precise on the inclusion age: X-18 Other types of resp ID: be more precise\nMethods: No date restriction? Is there a lower limit? Check to be inline with the PRISMA guidelines\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-253
|
https://f1000research.com/articles/12-252/v1
|
09 Mar 23
|
{
"type": "Systematic Review",
"title": "âAm I Masculine?â A metasynthesis of qualitative studies on traditional masculinity on infertility",
"authors": [
"Cennikon Pakpahan",
"Raditya Ibrahim",
"William William",
"Patricia S Kandar",
"Darmadi Darmadi",
"A. ST. Aldilah Khaerana",
"Supardi Supardi",
"Raditya Ibrahim",
"William William",
"Patricia S Kandar",
"Darmadi Darmadi",
"A. ST. Aldilah Khaerana",
"Supardi Supardi"
],
"abstract": "Background: The rate of infertility is increasing day by day. According to studies conducted worldwide, 30 million men are diagnosed with infertility. Cases of infertility are often associated with a failure to become male in society. Procreation and gender roles are often closely linked so that infertile men are often considered the second sex. Sometimes, this condition makes men question their masculinity. Methods: We performed a systematic review and metasynthesis with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guideline procedure on qualitative studies on ten databases exploring the experience of infertile men and their association with masculinity. Results: Twenty-four studies matched our question, and there are two major themes with eight subthemes that were obtained from the results of the metasynthesis of these studies. The impact of this gender issue is huge on menâs health and their social interactions. As a result, gender issues provide a space for debate and a burden on men. Sometimes, men develop mental health problems. The topic of masculinity and infertility is at odds with feminism and is susceptible to the societal stigma that results from the hegemonic conception of masculinity. Interestingly, the men must accept reality and follow the treatment process for infertility, although it affects their psychological well-being. Conclusions: These findings provide insight for physicians, as treating infertility requires a multidisciplinary team that does not only address procreation issues. Social issues related to gender roles often bring patients into harmful and dangerous conditions. To address the gender issue in men globally in several dimensions, however, a large study in various populations is still required.",
"keywords": [
"feminism",
"gender equality",
"infertility",
"masculinity",
"psychological well-being",
"metasynthesis",
"qualitative"
],
"content": "1. Introduction\n\nCreating a family is one of the reasons for people to get married. Newlywed couples are often expected to have children. But for those couples who take years to have children or even do not get the chance to, it can become an issue. Infertility for some couples can mean they have to make extra effort to get descendants. In some communities, it is undeniable that infertility has a significant impact on increasing number of divorce cases, economic difficulties, and even cases of losing the right to a funeral (Greil et al., 2010; Kumar & Singh, 2015). Infertility can be defined as the inability of a couple to become pregnant after 12 months of regular sexual intercourse without the use of contraception or protection (Zegers-Hochschild et al., 2009). Infertility affects 8%â12% of couples globally, and 20%â30% of cases are due to an issue with the male (Agarwal et al., 2015). It has been reported that there is a decline in the quality of male semen in several countries around the world (Dissanayake et al., 2019). Levine et al. reported that in the last 40 years, 1 in 20 men faced infertility problems (Levine et al., 2017). It is also confirmed by Agarwal et al. who said that at least 30 million men worldwide are confirmed infertile. The global rate of male infertility cases range from 2.5% to 12%. The highest numbers were found in Africa and Eastern Europe. This number indicates that infertility can occur anywhere (Agarwal et al., 2021).\n\nMen are reported to struggle when realizing that they are infertile. Feelings of vulnerability, isolation, failure, and futility are commonly found (Ravitsky & Kimmins, 2019). In some communities, it is undeniable that infertility has a significant impact on the increasing number of divorce cases, economic difficulties, and even cases of losing the right to a funeral (Greil et al., 2010; Kumar & Singh, 2015).\n\nThere is an intriguing relationship between the incidence of male infertility and its impact on men from a psychological and social perspective. These are inextricably linked to the persisting stigma surrounding infertility, which can lower a manâs self-esteem. This concept often means that infertile men are viewed as the âsecond sexâ or inferior masculinity. Infertile males are considered deviants based on hegemonic masculinity norms; infertility drives men to reconstruct and renegotiate their own masculine identity. This is influenced by the impact of hegemonic masculinity that is prevalent in parts of the world with different races and cultures (Burton, 2014).\n\nThe public often uses the term masculinity to define men as masculine while women are feminine. Masculinity has been viewed as strength, power, and aggressiveness and is formed in opposition to fear, anxiety, or weakness (Burton, 2014; Rotundi & Pitti, 2020). Men who have a muscular body and fulfill specific âcriteriaâ are frequently considered masculine. Being fertile is also considered a part of masculinity (Burton, 2014; Rivera & Scholar, 2020) This assumption often implies that masculinity is brought from birth.\n\nMany experts argue that masculinity is formed by the interaction of various factors. For example, historical and sociocultural factors, and geographic locationâand is not innate from birth (Rotundi & Pitti, 2020). According to the social constructivist theory of masculinity, gender is achieved through and by people and their environments. Nowadays, the ostensible boundary between gender (oneâs role in society) and sex (physiology reproductive function) is vanishing. Gender is something we perform in social interactions, not an innate part of us (Moynihan, 1998). Society tried to formulate the idea of masculinity based on the results of these interactions. Therefore, we have to be careful because it is possible that this viewpoint can lead to âhegemonic masculinity,â a condition where every man will race to be recognized as a ârealâ man (Connell & Messerschmidt, 2005).\n\nCurrently, it is undeniable that there has been a shift in society, especially in the population of young men. This community is more courageous in expressing their tenet socially coded, traditionally, as feminine (Anderson, 2018). More men are aware that masculinity is something they do and perform rather than what they are (Waling, 2019). The traditional masculinity concept argues that a man must be strong, not show many emotions, and must be independent and dominant. The virility means that a man can make a woman pregnant and function sexually. On the contrary, a male that does not fit these characteristics is not considered a real male. This act of overly understanding the traditional masculinity concept results in masculinity as toxic (Gannon et al., 2004; Rivera & Scholar, 2020).\n\nTraditional masculinity can limit personal growth for a man and can cause distress to those who do not meet those criteria. This can lead to depression, substance use, and even suicide (Gannon et al., 2004; Kupers, 2005). Traditional masculinity can also make a man feel shame for seeking help when in distress, mentally or physically, because of the image of being less masculine. The American Psychological Association (APA) argues that men who try to adapt to the traditional masculinity concept are more prone to have mental or physical problems (Gannon et al., 2004; Kupers, 2005). This underlies the APA to issue Guidelines for Psychological Practice with Boys and Men in August 2018. Boys raised in a traditional masculine household have a mindset that they must adopt this behavior to be accepted in society. Traditional masculinity can be found in everyday life, such as bullying, fighting, breaking the law, sexual assault, domestic violence, criminal acts, and drug use.\n\nFormerly, there are minimal social studies that explore the experience of infertile men accepting their roles from a gender perspective (Hanna & Gough, 2015). This is because reproductive biological function is identical to âfeminine area that demands female commitment and labour,â in part due to our gendered culture, yet âmale procreative contributions and reproductive masculinityâ are represented as unproblematic, with males being deemed fertile throughout their lifespan (Hanna & Gough, 2020).\n\nThere is a perception that success and masculinity go hand in hand, especially in Western myths. Having an illness related to sex roles, causes men to suppress their emotions (Moynihan, 1998). Social construction demands men to be fertile. This condition makes it very difficult for men to accept pain or express fears and weaknesses, most notably when they are dealing with infertility cases.\n\nInfertility as a medical issue and the significance of patriarchal authority are closely related. Androcentric medical knowledge constructs, legitimizes, and maintains gender norms, reflecting Western social and cultural notions of gender, in which rigid definitions of gender normativity are constructed, and any behavior or trait that exists outside these norms is considered deviant (Conrad & Barker, 2010).\n\nInfertile men lack these characteristics and are hence perceived as inadequate. As a result, infertility is a sign of a lack of masculinity because it does not fit within these ideals, labeling the person as less masculine (Dudgeon & Inhorn, 2003). Infertility poses a danger to masculinity because of the faulty belief that anything that exists outside the norms of masculinity must be attributed to femaleness, and thus males who break from these norms go closer to embodying female characteristics. In this sense, infertile males are connected with characteristics associated with femininity, such as being weaker and in need of assistance (Gannon et al., 2004). âMenâs sexuality is sanctioned and encouraged, whereas womenâs sexuality may be rigorously controlled, limited, and condemned,â according to a system of binary oppositions maintained by hegemonic masculine ideologies (Dudgeon & Inhorn, 2003).\n\nFinally, male infertility has lately been found to be perceived by men as a failure of manhood, a process fraught with stigma, silence, and suppression, as well as having a devastating and alienating effect on their life (Hanna & Gough, 2020).\n\nIn the following section, we try to investigate gender-based studies conducted on infertile men, providing an overview of the association between masculinity and male infertility. This study aims to explore the phenomenon of masculinity in cases of infertility\n\n\n2. Methods\n\nThe researchers/writers included the following studies: 1) qualitative studies using in-depth direct conversation or an online survey with the sample, 2) studies in English, 3) studies that contain information about gender issues, and 4) a study that was in a male infertility setting. Meanwhile, the researchers/writers excluded 1) literature review studies or other reviews, 2) quantitative studies, 3) studies that do not provide complete data such as information about gender issues, 4) Masculinity studies with qualitative methods but not on cases of infertility, 5) studies that are not in English, and 6) studies that are not available in full text.\n\nTo produce an adequate report on this systematic review, the researchers collected the data according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Figure 1) (Page et al., 2021). This study was registered on PROSPERO with registration number CRD42022327318.\n\nIn this systematic review, a study search was conducted in ten databases, such as PubMed, SAGE, Scopus, Taylor & Francis, Web of Science, ScienceDirect, Emerald, ProQuest, Wiley Online, and Springer Link. Other studies taken from sources (Google Scholar, researchsquare, medRix) that had not been mentioned earlier were also gathered. To search for studies involving the specific gender theme of âmasculinityâ in male infertility, the researchers used âgenderâ OR âmasculinityâ AND âmale infertilityâ as keywords. The study was presented qualitatively with regard to the information on gender phenomenon not according to the infertility problems they faced.\n\nTwo authors (CP and RI) conducted a separate study electronically using a previously approved keyword then used Mendeley to assess all the studies. The researchers (CI, RI, WW, and PSK) collected and retrieved the search and then filtered out the studies that fit the required theme. Any confusion that evolved during the search was discussed with the other authors (DD and SS).\n\nAfter the two authors (CP and RI) separated the studies according to Mendeley. CP, RI, WW, and PSK collected data together from studies that met the criteria. The researcher discussed the results/findings and then wrote the report. The year, author, location or population, sample size, title, and investigation results, including gender theme, were obtained from each study. If the required information was missed, we attempted to contact the articleâs corresponding author. We conducted a metasynthesis after collecting the data completely.\n\nThe critical appraisal skills program (CASP) was used to evaluate the quality of the studies that were included. This scale contained ten questions that evaluated the feasibility or quality of the included qualitative study. Each question would hold choices of âyes,â âno,â or âcanât tellâ (CASP, 2018). The assessment using this scale was only to evaluate whether the methodology of this study/article is appropriate or not. The assessment of this scale was carried out by two authors independently and then discussed the conclusion.\n\nThe researchers of this study conducted a review synthesis of the extracted journals. The synthesis consisted of two stages. The first stage was identifying the characteristics of the collected studies. We drew demographic data, research methods, and the number of samples used and then concluded the research results related to gender issues. The second stage was an in-depth review that was conducted using inductive phenomenology. At this stage, we explored descriptive information on how the experiences and interactions felt by the subjects comprehensively on whatever gender issue phenomenon they thought were related to the experience of daily awareness as infertile patients. The extracted data included perceptions (what the respondents hear and see), beliefs, memories, and feelings that are experienced every day. These data were synthesized from each of the included studies (Biggerstaff & Thompson, 2008). From this synthesis, the researchers emphasized what it means to be an infertile man for those with gender phenomena as part of the experience. Therefore, we focused on the results of in-depth interviews conducted by each author in their study.\n\nIn this second procedure, the researchers/writers identified texts that had certain expressions or meanings (meaning units), which are mapped into a particular theme and categorized based on the findings (thematic coding). At this stage, the researchers also removed the phrases that are not relevant to the goals of this study. Finally, each finding that had the same expression was grouped and then presented in the form of tables or diagrams.\n\n\n3. Results\n\nBased on the gender and masculinity theme, the researchers have tried to find evidence of the involvement of gender in cases of male infertility (Table 1). For the first topic, masculinity and male infertility, 24 studies were analyzed. All studies that were included for analysis were qualitative studies originating from Asia, Europe, Africa, and America. Some of these studies performed in-depth interviews in person, but some studies did online interviews. The quality of qualitative studies using the CASP form is carried out in all eligible journals. Table 2 presents the summary results of our study assessment.\n\nFrom the results, 24 studies discussed gender issues in male infertility. All studies are in the form of qualitative studies conducted through in-depth and online interviews. The study population was from various continents. The study was also conducted on men who had followed treatment up to the stage of assisted reproductive technology (ART). The theme of masculinity is a prominent issue in these studies, regardless of the country. Many of the men who experience infertility question their manhood. The researchers try to summarize some of the themes that they found concerning masculinity in men with infertility. There are two themes that were identified based on our analysis, and then we explore these themes in seven descriptive themes (Table 3).\n\nThe concept of gender phenomena in infertility is a topic that can cause friction. In generating offspring, collaboration between women and men is required. Infertility may be caused by one or both parties. Infertility is often associated with womenâs bodies, so if the cause is men, there is often excessive gender conflict either from the man himself or even from his environment.\n\nFrom the core analysis of all eligible studies, two major themes surround the âgender issueâ central phenomenon in infertility cases. The two big themes are:\n\n1. Personal perceptions and beliefs\n\n2. Sociocultural norms, values, and expectations\n\nThese two main themes are phenomena experienced by infertile men related to masculinity. The researchers/writers synthesize the menâs experiences again on these two major themes. These themes were analyzed in describing the experiences that men felt about being infertile. The researchers/writers summarize the results and the studies involved in describing this phenomenon (Table 3).\n\n\n4. Discussion\n\nWe are faced with a situation where infertility is stigmatized and it is challenging to identify cases of it because of this (Hinton & Miller, 2013). This phenomenon has not changed much in recent decades. Some communities still label infertility as a failure that makes men reluctant to be treated. Sometimes, men who struggle with infertility are often excluded from the community and considered as a âsecond sexâ (Rimer, 2010).\n\nBased on the data synthesis in this study, two significant themes underlie gender issues in infertility cases. But both influence each other so that it harms menâs acceptance of themselves, especially in handling infertility cases. Some factors come from the man himself, and some influences are from society. The findings of this study indicate that the existence of social norms such as masculinity and stigma have an impact on menâs perceptions of infertility. Many respondents from the research revealed that infertility makes them feel they are not men. This is because the patriarchal system is still very prominent across the globe. Likewise, this binary gender and sex system is still dominantly accepted throughout the world. There is a culture that thinks hegemonic masculinity is considered as an appropriate construction to define men. It is claimed that any shift in value from this construction is considered a decrease in masculinity. These demands are not only obtained by men from the norms or surrounding culture but can also come from their understanding of religion or belief (Arya & Dibb, 2016; Tabong & Adongo, 2013). Even some men experience their condition being underestimated by their wives (Gannon et al., 2004). This condition is what the researchers/writers consider to be traditional masculinities.\n\nTraditional masculinities have a major negative impact on menâs willingness to share their situation with others. Because they do not want to be perceived as weak or less masculine, men are reluctant to consult with and seek assistance. The infertile men also face a dilemma when seeking help. Although Assisted Reproduction Technology is often provided to assist men, it actually becomes a burden for them. Apart from financial concerns, strong religious values also often put them in a difficult position (Birenbaum-Carmeli & Inhorn, 2009). Even though the demands are still targeted at them, a man has his own value when he has biological children. In some countries, being a man automatically means one becomes a father. For example, in the Middle East, fatherhood is traditionally considered the equivalent of manhood. Men are viewed as powerful figures when they participate in reproduction. So, men will feel threatened when they are unable to reproduce (Inhorn, 2004).\n\nThis can lead them to severe anxiety and depression (Bahar et al., 2019). This is the dangerous impact of traditional masculinities on infertile men, a condition that can eventually be harmful to the man.\n\nYang et al. conducted a study in China reporting the uncertain prevalence of infertile men who experienced depression. Depression (20.8%), anxiety (7.8%), and a combination of the two (15.5$) were present in men, respectively (Yang et al., 2017). Our findings also suggest that infertile men are significantly at risk for depression and anxiety disorders. The disorder may be brought on by the demands of gender itself or by other factors.\n\nThere are no definite reports on whether this gender role is needed to have the same impact on men. Some assumed an association between gender role expectation and mental disorders in men (Ahmadi et al., 2011; Folkvord et al., 2005). In addition, one of the main factors contributing to males developing mental disorders is the costs they incur for treatment (Bai et al., 2019).\n\nMen with fertility disturbances are often labeled as men with a âlack of sexuality.â They are considered not to have good sexual performance (Pattnaik et al., 2016). Although they differ, there is a connection between male infertility and sexual function. It is not uncommon for male infertility to cause a sexual dysfunction experience. Measurements using the International Index of Erectile Function score showed a correlation between infertility and the incidence of erectile dysfunction (Coward et al., 2019; Gao et al., 2013; Ozkan et al., 2015; Sahin et al., 2017). Patients with primary male infertility are more likely to have symptoms of erectile dysfunction than secondary male infertility (Sahin et al., 2017). Various medical procedures undertaken during the pregnancy program itself can also alter sexual behavior and the couple intimacy, often leading to temporary sexual dysfunction (Bechoua et al., 2016). Additionally, sexual satisfaction can also be reduced due to the forced sexual activity for conception purposes (Tao et al., 2011).\n\nEjaculatory disorders have also been reported in several studies, aside from erectile dysfunction (Gao et al., 2013). This disorder can also be a part of the anxiety experienced by infertile men. This variable is intriguing because it can lead to infertility due to sexual function disorders (Yang et al., 2017), but this complaint may arise after being diagnosed with infertility. However, the two can be directly or indirectly related. In the community, impaired sexual function is often also considered a sign of DE-masculinization (Pakpahan et al., 2021). There is a very complex and interconnected relationship between male infertility, sexual dysfunction, depression, and anxiety (Figure 2).\n\nMale infertility causes men to be stigmatized as failed men because of the masculinity demands. On the other hand, infertile men are also sometimes caused by sexual dysfunction which is often identified as men's lack of masculinity. These two conditions affect their mentality. Men with mental disorders, especially depression and anxiety, also get the label lack of masculinity.\n\nThe World Health Organization states that our sociocultural circumstances still often demand that men do not get sick, especially those with mental illness (Gough & Novikova, 2020). This is what makes men often hide their real condition. Problems will be complicated when they are infertile and depressed. Infertile men with depression and anxiety face a double burden of their condition. Their emotions and mentality as men are also questioned in the community (Gourounti et al., 2010).\n\nMale infertility is not a condition that threatens menâs life, but because of the stigma and social punishment that come with it, men are put under a great pressure. This is especially in a patriarchal culture with social and religious values that are not easy to control. These two things are values that have been passed down from generation to generation and are firmly attached to the community. These values are like big strongholds in the community that are not easily torn down. It seems that there is a strong association between the âmasculinity issueâ and the way men face infertility.\n\nThere is a phenomenon in a society that a personâs masculinity will affect health behavior. This phenomenon is not always true. Rochelle reports that norms of masculinity do not always go hand in hand with a personâs healthy behavior. Instead, Rochelle claims that a personâs support system influences a personâs healthy behavior (Rochelle, 2020). In the case of male infertility, couplesâ therapy and family education are very important to change the perspective of men who are infertile. If the patientâs support system is warm, feelings of DE-masculinization will not occur.\n\nChanging perceptions and social constructions is not easy but requires attention. It is important to involve community organizations and religious leaders in creating the emotional dangers of social construction. Traditional masculinities put men as the victims. Men have the right to be independent of their conditions without being judged by the norms and values that isolate them. Men confiding in fellow men about infertility is beneficial. or others. Almost certainly fellow men better understand the conditions and emotions of each other. Men with infertility need a place to share and exchange ideas about their problems such as support groups.\n\nThe media has played a big role in changing any culture in this century. The âMeToo movement,â a massive American movement against sexual harassment, has hugely impacted twenty-first-century culture. Against âtraditional masculinities campaignâ should also need a platform and place like this to elevate menâs self-confidence. Men have the right to speak for themselves if it does not destroy others. According to Knudsen and Andersen, one of the genres where values of masculinity are particularly obvious and can be represented is advertising or campaigns that are employed as vehicles of cultural myth. The movement against traditional masculinities requires the media to convey the detrimental impact of this phenomenon. Lynxâs Is It Ok for Guys (2017) and Gilletteâs The Best a Man Can Be (2019) are two campaigns that have asserted no to overestimating masculinity with different audiences. Although it has received negative criticism, consistent ideas like these need to be communicated (Knudsen & Andersen, 2020). In 2021, Jane Campionâs The Power of the Dog was also appreciated for trying to dispel the toxicity of excessive masculinity. We need movie ideas that depict dangerous extreme masculinity, especially in the case of infertility (so far, not many films have told). Cervi and Knights argue that a combination of social, medical, and organizational perspectives that are heterogeneous and engaged in reproduction is needed for the treatment of infertility. All of them can play a role in providing medication and nutrition, providing legal advice or moral therapy to relieve stress (Cervi & Knights, 2022). Thus, this solution is required to fight the toxicity of excessive masculinity.\n\nThere are many possibilities of finding contradictions between feminism and counter-traditional masculinity in couples who are dealing with infertility, especially if the male causes the infertility. One very interesting fact related to infertility is that although the main cause is men, the focus is women. Even women bear the severe consequences of infertility treatment, for example, ART (Assisted Reproduction Technology) (Hanna & Gough, 2020). When couples must choose ART, at this stage, women are required to undergo hormonal stimulation that they should not experience. Hormonal stimulation is known to have very agonizing effects for a womanâs body, and it can even be life-threatening (Mahajan, 2013). Because of the failure of male reproductive function, women must bear the consequences. Sometimes, fertile women take this path to maintain monogamous marriages and obtain offspring (Lorber, 1989).\n\nOne of the interesting proposals from Hanna and Gough (2015) is to involve men more openly in reproductive research. This will have practical implications for service delivery. It is necessary to provide men a voice and support them to connect the needs of men and the management of infertility by men (Hanna & Gough, 2015).\n\nAbove all, we need to understand that the movement against traditional masculinity is beneficial for all of society. Traditional masculinity is a very dangerous condition not only for men but also for women and children. Traditional masculinity is actually an over-glorification of men that causes negative effects on others (Sculos, 2017).\n\nThis study is still limited to synthesizing the experience of phenomena that individuals feel about infertile men. But a more profound grounded theory of why infertility must be synchronized with masculinity is still needed. In addition, issues close to society and culture are still limited to a few populations and not globally, so the generalization of masculinity with infertility is still not evenly distributed. Similar studies with varied individuals and cultures still need to be conducted again for a global masculine phenomenon. Moreover, some of the studies that we include in this review do not explicitly explain the number of samples included and the process of obtaining the data.\n\n\n5. Conclusion\n\nInfertility is a common condition. Modernization and public awareness have encouraged the knowledge of this condition, although there are still hidden cases is often caused by cultural and social views that infertility is humiliation, especially if the male is the main problem. In a patriarchal culture, male infertility cause is considered a failure in men. This is frequently a result of societyâs overly high expectations for men. This unwarranted expectation arises because of the over-glorification of men in the society, even though men are the same as women who both have limitations.\n\nTraditional masculinities pose a huge threat to men. The feeling of âisolation, loneliness, powerlessness, and recklessnessâ even depression and other psychological disorders are real impacts for men with infertility. We must realize that against traditional masculinity does not mean threatening the feminist movement. There is nothing to do to fight traditional masculinity toward feminism. Instead, against traditional masculinity helps feminism find its place.",
"appendix": "Data availability\n\nFigshare: PRISMA_2020_checklist Masculinity.docx. https://doi.org/10.6084/m9.figshare.22193602.v1 (Pakpahan, 2023).\n\nThis project contains the following extended data:\n\nâ Figure 1 Prisma.doc\n\nPRISMA checklist for âAm I Masculine?â A metasynthesis of qualitative studies on traditional masculinity on infertility. https://doi.org/10.6084/m9.figshare.22193602.v1 (Pakpahan, 2023).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAgarwal A, Baskaran S, Parekh N, et al.: Male infertility. Lancet. 2021; 397(10271): 319â333. Publisher Full Text\n\nAgarwal A, Mulgund A, Hamada A, et al.: A unique view on male infertility around the globe. Reprod. Biol. Endocrinol. 2015; 13(1): 37â39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAhmadi H, Montaser-Kouhsari L, Nowroozi MR, et al.: Male infertility and depression: a neglected problem in the middle east. J. Sex. 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}
|
[
{
"id": "166012",
"date": "20 Mar 2023",
"name": "Abiodun Mathias Emokpae",
"expertise": [
"Reviewer Expertise Reproductive Biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study examines the effect of traditional masculinity on male infertility. This perception varied in size and shape from one society to another, but all geared towards discriminating against the infertile man. The article is well written and has contributed to knowledge in male infertility study. However, the keywords should be derived using âMeSH on Demandâ.\nThe association of male infertility with masculinity is inappropriate and it makes infertile men regard themselves as inadequate, incomplete and less masculine. The disorder therefore, poses danger to masculinity because of wrong perception and/or belief. The perception of inability to achieve pregnancy as failure of manhood is misleading and has contributed to several associated psychological and adverse health effects. Infertility can be due to male or female gender, therefore requires cooperation and patience by the couples. It should not be stigmatized and affected men should be encouraged to present themselves for evaluation and treatment. Open discussion against traditional masculinity may be beneficial to solving several psychological problems associated with male infertility.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "9515",
"date": "03 Apr 2023",
"name": "Cennikon Pakpahan",
"role": "Author Response",
"response": "Dear Emokpae, Thank you very much for your comments on our article, We will revise it according to your suggestion. Regards"
}
]
},
{
"id": "166017",
"date": "28 Mar 2023",
"name": "May Soe Aung",
"expertise": [
"Reviewer Expertise Maternal Health",
"Reproductive Health",
"Infection control measures",
"One Health Approach"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article addresses the linkage of masculinity, infertility and mental health consequences by means of metasynthesis based on qualitative studies. The authors can explore the important points in the abstract. Methodology is sound and discussion is comprehensive including good description about limitations based on the study findings. Some issues that need to be modified are as follows:\nIntroduction:\nMental health problems like anxiety and depression should be added because these are highlighted in the discussion.\n\nAlthough the detail of the masculinity and infertility are well introduced, more justifiable points with reasons linking to the aim of the study are recommended.\n\nRepetition of third sentence from the first paragraph in the second paragraph should be modified.\n\nIn section 1.1, fifth sentence by Moynihan is suggested to be rewritten to avoid direct copy of the original format.\n\nFlow Diagram:\n\nIt is needed to check and confirm why the number of studies after duplicates removed (n=145) is lower than that after screening (n=242).\n\nMethods:\nIt is suggested to mention the search tool used to identify the components of the research question such as SPIDER (Cooke et al. 20121).\n\nIt is better to mention whether the authors searched for gray literatures and doctoral dissertations.\n\nRange of years should be mentioned in the inclusion criteria of the studies.\n\nFor enhancing transparency in reporting the synthesis of qualitative research, the ENTREO statement is recommended. However, the authors can provide the justification for preferring PRISMA checklist.\n\nResults: For Table 1:\n\nIt will be more suitable to order the included studies according to the year of study.\n\nColor using for author name should be consistent; because that of no. 6 is different from others.\n\nUnder Sample Size in no.12 and 15, threads, messages, posts, unique posters are suggested to clarify for understanding of readers because the data collection method mentioned in the inclusion criteria is in-depth interview.\n\nIt will be better to modify as âStudy areaâ in place of âPopulationâ.\n\nIn the last column, mentioning âResults of investigationâ is enough and so explanation within parentheses are suggested to remove.\nDiscussion: In the fourth paragraph, first line should be written with description instead of âThisâ at the start. Conclusion: The contents of the conclusion in the abstract and the body of manuscript should be consistent.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes",
"responses": [
{
"c_id": "9516",
"date": "03 Apr 2023",
"name": "Cennikon Pakpahan",
"role": "Author Response",
"response": "Dear Soe Aung, Thank you for the comments on our article, it means a lot to us. We will try to revise it according to the reviewer's comments. Regards"
}
]
},
{
"id": "166016",
"date": "28 Mar 2023",
"name": "Suriyani Muhamad",
"expertise": [
"Reviewer Expertise overall",
"it can be understood",
"except for medical and specific terms of medical"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI can say that this review paper offered a different perspective on the male infertility issue by integrating the masculinity aspect.\n\nIt is a great review paper that adds to the present review papers on male infertility studies.\n\nI suggest elaborating more on the below paragraph, perhaps with an example:\n\n\"Above all, we need to understand that the movement against traditional masculinity is beneficial for all of society. Traditional masculinity is a very dangerous condition not only for men but also for women and children. Traditional masculinity is actually an over-glorification of men that causes negative effects on others (Sculos, 2017).\"\n\nOverall, this review paper is recommended to be indexed.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly",
"responses": [
{
"c_id": "9514",
"date": "03 Apr 2023",
"name": "Cennikon Pakpahan",
"role": "Author Response",
"response": "Dear Muhammad, Thank you very much for the comments given on our article. We will update according to the reviewer's comment. Regards, Author"
}
]
}
] | 1
|
https://f1000research.com/articles/12-252
|
https://f1000research.com/articles/12-249/v1
|
07 Mar 23
|
{
"type": "Method Article",
"title": "Evidence-based operational model for conducting an online, synchronous, proctored, closed book professional examination during the COVID-19 pandemic lockdown",
"authors": [
"Jaiprakash Mohanraj",
"Ivan Rolland Karkada",
"Rusli Nordin",
"Jaiprakash Mohanraj",
"Rusli Nordin"
],
"abstract": "Background: Medical education has migrated online as a result of the COVID-19 pandemic. Formative and summative evaluation are critical in medical education to support the learning process. However, many problems occur during online assessment, such as internet access, proctoring, and reliable software. To mitigate this situation, there is presently no standardised model available that demonstrates reliability and validity, particularly when it involves high-stake examinations. The goal of this study was to introduce an evidence-based operational model that was used to conduct a comprehensive high stake online examination to Year 2 MBBS students during the COVID-19 pandemic. Prior to entering the clinical years (year 3-5), year 2 MBBS students were expected to pass their high-stake examination earlier conducted as face-to-face session which is now administered online. Methods: The model proposed enables the conduct of an online, synchronous, proctored, closed-book examination in real-time that is comparable to the face-to-face (FTF) manual examination. The examination and invigilation were carried out using the Learning Management System (LMS) and Google Meet applications with the existing information technology (IT) facilities and the Standard Operating Procedure (SOP) was developed to optimise and integrate the technology effectively. Results: Both students and faculty were satisfied with the online examination, with the latter being more so. The online examination with a Cronbach alpha score of 0.71 suggested good internal consistency and reliability and various test of validity ensured this online model was dependable, reproducible and concurrent with other examinations. Conclusions: We concluded that this operational model is a credible alternative to the FTF examination that requires new learning technology and is very cost effective. Thus, we highly recommend this model for all examinations that involve participation of off campus students.",
"keywords": [
"Online Examination",
"Synchronous",
"Proctored",
"Closed Book Examination",
"Assessment"
],
"content": "Introduction\n\nAssessments are an essential element of medical education, and when combined with subsequent feedback on students' performance, they serve a critical role in assisting students in improving their learning (Preston et al., 2020). As Bransford et al. point out, assessment is a critical component of effective learning; thus, formative and summative assessment constitute one of the many important components of formal education (Muzaffar et al., 2020, Bransford et al., 1999). The authors indicate that teaching and learning processes need to be assessment-centred to provide learners with opportunities to demonstrate their developing abilities and receive support to enhance their learning. This is especially true for any examination with a high-stake component. Under these circumstances, the examination department's responsibility in conducting a fair and trustworthy examination in a conducive atmosphere becomes critical in ensuring that the program's integrity and quality are maintained through the administration of high quality, peer reviewed examinations.\n\nWith emerging technological advancements, the challenges in conducting high-stake examinations are growing, and new challenges have especially emerged during the COVID-19 pandemic in 2020. During these trying times, governments-imposed lockdowns and urged universities to deliver their curriculum using online platforms to continue studentsâ progress. More than delivering the teaching and learning content, the challenge was enormous in assessing student learning. This is because assessment drives learning and the need for valid and reliable online assessment tools cannot be overemphasized (Reyna, 2020).\n\nBefore the COVID-19 pandemic, medical institutions frequently conducted formative assessments using the Learning Management System (LMS) platform as the main mode to assess learning, but all summative examinations, especially high-stake examinations, were conducted face-to-face (FTF). The written paper, which included the Multiple Choice Questions (MCQ), Modified Essay Questions (MEQ), Case-Based Questions (CBQ) and Problem-Based Questions (PBQ) were conducted in the examination hall, Objective Structured Practical Examinations (OSPE) were conducted either as mobile stations or using a digital platform, Objective Structured Clinical Examinations (OSCE) were conducted as clinical stations using mannequins and simulated patients and Clinical Examinations that include long and short cases were conducted in hospitals or clinical settings. All these examinations were proctored in FTF sessions. Although many universities across the globe used assignments as their summative assessment tool in their online distance learning programs, the use of assignments in assessing a medical graduate program has been close to nil. In the current COVID-19 pandemic, online being the only mode of assessment possible, the challenge to conduct all these examinations synchronously and proctored has been a daunting task. As many teaching and learning methods were vastly prescribed, practically no guidelines were available in conducting online summative examinations that are proctored, synchronous and conducted with an acceptable degree of validity and reliability. Thus, the aim of this research was to conduct online, synchronous, proctors & closed book exam that was reliable and valid. The objectives of this study were to (1) Perform needs analysis to identify the strengths, weakness, opportunities and threat in conducting this exam. (2) To develop and design SOP and systems that will enable the conduct of this exam. (3) To conduct a high-stake professional exam using the developed and tested Exam system. (4) To evaluate the reliability and validity of this system.\n\nIn this paper, we present an operational model that was adapted in a medical and allied health university to conduct online, proctored and synchronous high-stake barrier examinations in the First Professional Examination in Year 2, and the Final Professional Examination in Year 5 of the 5-year medical (MBBS) program. We used innovative approaches using existing online tools and platforms to conduct these examinations. Our aim was to conduct an online, synchronous, proctored, high-stake summative examination that has a high degree of validity and reliability.\n\n\nMethods\n\nThe proposed system is an examination process based on the Modified Fish Bone Online Examination Model (MFBOEM) (Figure 1).\n\nThis examination model suite is designed for a scenario in which students can take an ONLINE test anywhere in the globe, but the examination is conducted and controlled in the university's information technology laboratory by a team of examination committee experts, qualified invigilators, and a technical team This examination was conducted in the school of medicine for Year MBBS students in August 2022. The research, development, implementation, and analysis of this system was conducted in the period between June to October 2022.\n\nBased on the universityâs prescribed examination regulation and statutory approval by the relevant university stakeholder committees (Senate/Faculty Board/Assessment & Examination Board), the Standard Operating Procedure (SOP) for the conduct of the proctored online examination protocol was adopted (Figure 1). In line with the examination regulations and SOP, an endorsed comprehensive timetable depicting chronological events starting from Step 1 to Step 9 leading towards completion of the online proctored examination was developed. The stipulated events are summarized in a modified fish bone diagram starting with the standard set guidelines in the left 3 boxes running through the parallel events for invigilators and students finally leading to effective end results as shown in Figure 1.\n\nThe process of the online exam is described in 9 steps for easy understanding and implementation.\n\nSTEP 1: Initial communication\n\nStudents and staff were briefed through online google meet platform regarding the examination regulation and SOP for preparation and implementation of the online examination. During this session, students were also made aware of all the eligibility criteria for the ONLINE examination as indicated in Step 2 in Figure 1.\n\nSTEP 2: Requirements\n\nImplementation of the ONLINE examination requires both hardware and software items that cater for all stakeholders (students, invigilators and IT laboratory). Requirement fulfilment and eligibility adherence of students were assessed using pre-determined Google survey forms, and further follow-up from Step 1 briefing studentâs administrative shortfall (registration/ID/fees etc.) were addressed by the Year Coordinator. The forms were used to collect information regarding students concerns with regard to the online exam. The data was not used for any documentation or analysis in the exam or in this article. This form was circulated 2 weeks before the mock exam. Hardware and software issues were addressed by the relevant technical team.\n\nLikewise, invigilator requirements (such as access to Google Meet & LMS accounts) were appraised and issues, if any, were ameliorated by one-to-one consultation with each invigilator with the help of an in-house technical team.\n\nTechnical requirements:\n\n1. Examination Venue: IT Lab 1A and 1B with the capacity of 50 desktop Personal Computers (PCs) in each laboratory.\n\n2. Operating System: Windows edition 10 Home Single Language.\n\n3. System: Desktop PC setup with earphones.\n\n4. Processor: Intel(R) Core (TM) i506400CPU@2.70GHz 2.71Ghz, 4GB RAM & 64-bit operating system.\n\n5. Internet Specification: A minimum speed of 500 Mbps through LAN cable.\n\n6. Software Specifications:\n\na. Google Meet: A video-communication portal developed by Google (G-suite education license, compatible on android and iOS platforms for online streaming, recording and storage.\n\nb. Learning Management System (LMS): Moodle 3.7 version, managed by an internal professional team.\n\nNon-technical requirements:\n\n1. Invigilatorsâ requirements:\n\na. Invigilators must have access to a university allotted Google Meet account.\n\nb. Invigilators must have access to universityâs LMS account.\n\n2. Studentsâ requirements: (eligibility criteria)\n\na. Examination registration (attendance, fees clearance, identification card, etc.).\n\nb. Laptop/desktop (with camera enabled).\n\nc. Internet data pack of a minimum 10GB (unlimited data pack was recommended).\n\nd. Access to a university allotted online Google student account for Google meet.\n\ne. Access to university allotted online LMS.\n\nAfter the provisional approval of processes of online examination design plan, it was thoroughly tested by the exam and IT technical team for its technical viability, credibility and reliability, following which the final version of this exam model was approved by the relevant university boards.\n\nSTEP 3: Testing the system\n\nStudents were briefed on all enquiries based on their feedback from Step 2. Students were also instructed on the conduct of the Mock Online Examination.\n\nPattern of examination:\n\n⢠Use of LMS as the examination tool.\n\n⢠Proctoring was done by live visual streaming of students taking their examination by desktop/laptop and mobile devices through Google Meet with simultaneous window display of the question paper in the LMS platform.\n\n⢠Question components included single best answer, short essay question (SEQ), and case-based question (CBQ).\n\n⢠Duration: Each Single Response Answer (SRA) was allotted 90 seconds, SEQ/CBQ â 15 minutes per question.\n\n⢠The response method included checking the correct box for the options given under SRA and typing the answer in the text box provided for SEQ/CBQ.\n\n⢠Examination rules, regulations and SOPs were posted as self-read material on a specific LMS examination folder, which was made available to both staff and students.\n\nTraining of invigilators:\n\nThis process started with briefing on the practical training followed by hands-on experience in developing the necessary skills in the software and all the relevant platforms. The training was to familiarize the use of Google Meet, such as opening an account, creating single and multiple Google Meet session links with association of time in the Google Calendar, recording of Google session links, setting up of single and multiple links audio & video communication with students, sharing of these URLs links via email to students, and extraction of the recorded video of the Google session to the database. All invigilators were familiar with the use of LMS for examination purposes as these were used frequently during the teaching-learning activities.\n\nSTEP 4: Implementation of the mock exam\n\nAfter establishing the Google Meet connection with the invigilators, the students were requested to share their entire screen with the invigilators. Upon approval from the invigilators, students were instructed to open the examination folder in the LMS. Following instruction from the invigilators, students commenced reading the examination regulations and SOP for the online examination. At the stipulated time, students were asked to open the examination question paper using a password provided individually on studentsâ WhatsApp. At the conclusion of the examination, students cross checked to confirm that all questions have been attempted and, with the permission of the invigilators, ensured that the submit button had been clicked to submit the answers and exit the platforms, thus marking the end of the examinations.\n\nIT Lab Workstation Design Plan: Each invigilator was accommodated in a climate controlled, ergonomically structured IT-Lab observing a physical distance of one meter. As shown in Figure 2, individual invigilators were assigned an internet (LAN connection) enabled two (2) desktop PCs and two (2) headphone sets (ear lobe covered). Each PC had three studentsâ Google Meet link browsers with full screen shared depicting LMS page downsized to fit the PC screen. Thus, individual invigilators have in total six studentsâ Google Meet links, three each in two PCs. All information were stored in a secure server and later retrieved at the data centre as and when necessary.\n\nQuestion paper setting in LMS:\n\n1. Examination question papers were made available in a specially created examination folder in the LMS.\n\n2. Question papers had pre-determined starting and ending times (after ending time, students could no longer attempt the questions).\n\n3. Students were allowed to scroll forward and backward in the question grid (i.e., they didnât have to go by a sequential order but on a random basis.\n\n4. Questions and the distractor options are shuffled for each student.\n\n5. LMS setting ensured that the question paper cannot be downloaded.\n\n6. Students have to key in answers in the question paper in the space provided in the LMS.\n\nInvigilation Mode: According to the time scheduled in the examination timetable, the invigilators reported to the venue and Chief Examination Invigilator for the day, and took charge of their examination workstation as per the duty allocation by the examination team and confirmed the workstation readiness as per the training instructions. Based on the student list allotment for each invigilator, which is pre-determined by the examination team (one invigilator is assigned to six students), the invigilator sent a separate student-specific Google Meet link for every student through email. The students logged in using this link and established the connection with the invigilator 30 minutes in advance of actual examination starting time. This was to ensure that any technical issues are sorted before the start of the examination. Invigilators then informed the students regarding the video recording of the session and started recording the Google Meet session followed by preparation of the student as per the prescribed SOPs as summarized below.\n\n1. Students will display their ID cards/Examination hall tickets.\n\n2. Students will show their surrounding (360 degrees) area including the desk through desktop/laptop camera by rotation of the camera to confirm that there is no reading materials/other electronic devices (apart from the permissible mobile phone) or any other persons in the space identified by the student taking the examination (Figure 3 Studentâs Work Station). Students are informed that the invigilators are at the liberty to make such inspection at any point of time when there is suspicion aroused by the studentâs activity.\n\n3. Students are directed to arrange their seating positions such that they face the wall on two sides (Figure 3).\n\n4. Students are directed to place their mobile phone with camera turned on and connected all through the examination on to the wall on their left or right side depending on their seating arrangements (Figures 3 and 4).\n\n5. Students are advised to keep their session audio unmuted throughout the examination period.\n\n6. Students will now be directed to open the LMS in another browser, and share this browser with the invigilator and open the examination folder and start reading the examination hall regulation and SOP for online examination under the supervision of the invigilator for 30 minutes.\n\n7. Students will now be directed to open the online question paper as per the allotted examination starting time with the password for the question paper shared to individual students five minutes before the examination starting time.\n\n8. Students will be advised to start attempting the question paper in real time. They are also reminded that the question paper is time bound (i.e., it will open and close at a pre-determined time setting), and no downloading is allowed.\n\n9. Six students will be proctored by one invigilator for the entire period of the examination.\n\n10. Students will be advised to raise their hand (physically) in front of the camera in case any trouble shooting is required to notify the concerned invigilator. The invigilator would then assist them individually without disturbing the other students (mute all other students except the one with issues) as each student is connected to the invigilator with specific link to avoid overlap.\n\n11. Students are advised to contact their invigilator immediately through their WhatsApp video call/normal call in case they have issues with their PCs/internet connection. They are also advised to be in touch with their invigilator until the issues are addressed by the examination/technical team.\n\n12. Students are advised that, as per the online examination rules, if the student loses internet connectivity or experiences any other technical issues that cannot be rectified within 15 minutes, the student will have to take the same exam on another day.\n\n13. Students who wish to use the washroom will have to notify the invigilator, as explained in Step 8 above.\n\n14. Students are advised to focus on the screen as much as possible with reasonable humane excuses, but if the invigilator detects suspicious eye movement of the student, the invigilator can ask the student to display the surrounding, as explained in Step 2 with warning, and three such recorded warnings can be considered as exam irregularity.\n\n15. Students are notified of the last 15 minutes before examination ending time.\n\n16. Students are directed to click on the submit button in the LMS page after they have completed their examination.\n\n17. Students are advised to exit first LMS and then Google Meet session when the session recording stops.\n\nIn the event that the invigilator needs to leave his\\her workstation, the invigilator must notify the Chief Examination Invigilator for the day so that a substitute invigilator could be arranged for that period. The invigilator will end the session after affirming that all events have been completed and send the recorded video link for each student for documentation and storage to the Chief Examination Invigilator.\n\nInvigilators will be required to sign the Examination Oath Declaration form before commencement of the examination activity. The Chief Examination Invigilator, together with the Examination Coordinator and Examination Team, will supervise the conduct of the examination.\n\nSecurity check: The invigilators were instructed to check for screen and cursor movements. The movements observed on the screen were expected to be active indicating the active use of the cursor. The signals to indicate full screen sharing/student eye movement/LMS and Google Meet log were stringently observed thus providing evidence on log activity during the examination time for each student.\n\nSTEP 5: Student mock/trial examination and proctored online examination invigilation feedback\n\nAfter completing their mock examinations, students were encouraged to complete the pre-determined Google Survey Form to share their experiences, difficulties and any suggestions for improvement. The Google Survey form can be found under Extended data (Mohanraj, 2022).\n\nSimilarly, after completing the online mock examinations, invigilators are encouraged to complete the pre-determined Google Survey Form to share their experience and difficulties, how they handled issues (if any) and finally, any suggestions for improvement.\n\nSeparately designed Google Survey Form links are distributed via email to both students and invigilators.\n\nSTEP 6: Studentsâ and invigilatorsâ briefing on post mock/trial examination (report from feedback 1)\n\nStudents were briefed (arranged by online Google Meet session) on the overall conduct of the mock examinations (doâs and donâts emerging from the mock examinations) in line with the invigilatorsâ inputs of proctoring students sensitized with technical short falls observed (if any) during the examination, technical guidelines, and steps to improve the situation, were recommended. Concerns raised from the studentsâ feedback will be looked into and addressed accordingly.\n\nInvigilators were also briefed (arranged by online Google Meet session for briefing) on the overall conduct of the mock examinations. Studentsâ feedback on the examination conducted and proctoring issues (if any) were raised, addressed and rectified. Concerns raised from invigilatorsâ feedback were shared among all invigilators and solutions discussed.\n\nTaking into account studentsâ and staffâs feedback on the two mock examinations, the examination department will address and correct all issues. During the Professional Examination, no technical issues were encountered.\n\nSTEP 7: Studentsâ and invigilatorsâ briefing on the final proctored online examination\n\nStudents were reminded of the Examination Regulation and SOPs for the final online examination. Students were made aware of the changes incorporated into the examination conduct based on feedback and asked to adhere to the dress code during the online examination as per the examination rules and regulations. Invigilators were briefed on the overall proctoring guidelines and changes incorporated thereof, based on the experience and inputs from studentsâ and invigilatorsâ feedback from the trial runs of the mock examinations.\n\nSTEP 8: Student final examination and proctored online examination invigilation\n\nAccording to the time scheduled in the examination timetable, upon fulfilment of all online examination prescribed requirements, the final online examination was conducted as explained in step. 4 and with all ground experience endured and considering the feedback from both students and invigilators during the trial mock examination.\n\nThe invigilators will check with the Chief Examination Invigilator, who will ensure that all students have attempted and submitted their examination answers correctly in the LMS by cross-checking with the technical team (LMS) before releasing the students. The Chief Examination Invigilator, together with the invigilators, will compile the invigilation report at the end of the examination, to be submitted to the Examination Unit.\n\nSTEP 9: Student final exam and proctored online examination invigilation feedback\n\nStudents and invigilators, after completing the proctored online final examinations, are encouraged to complete the pre-determined Google Survey Form to share their experiences, difficulties and any suggestions for improvement. Separately designed Google Survey Form links are distributed via email to both students and invigilators. All feedback from both students and invigilators are processed and analysed. Output from the analysis paves the way towards amending the online examination regulation SOPs. This marks the end of the proctored online examination process.\n\nOur process has been tested to address the possibility of malpractice by using the following approach:\n\n1. Use of two video cameras: The use of video feed from the studentâs phone and laptop gave us a wide angle to view his/her working space. This prevented the student from using any paper or technology-based information as a malpractice tool.\n\n2. Monitoring studentâs eye movement and keyboard activities: The use of split screens ensured that every time the student looked away from the screen, he/she was expected to look down to type his/her answers. Whenever the studentâs keyboard was in use, the content typed would be visible to the invigilator to view on the LMS Examination page. The invigilator monitored the studentâs gaze and also scrutinized his/her keyboard activity. Appropriate warning would be given if change in his/her point of gaze was observed and/or if the keyboard was found idle for a considerable amount of time to raise any suspicion of malpractice.\n\n3. Preventing third party verbal assistance by using the sound system: The students were instructed to keep their microphones on their devices on at all times. This was to ensure that there was no verbal assistance from anyone in the vicinity of the candidate.\n\n4. Preventing use of digital or paper-based aids by using desk and surrounding check protocols: The students were instructed that anytime the invigilatorâs suspicion was credibly aroused, they would be requested to use their camera to show their desk space and the surrounding environment. This would improve the invigilators confidence and freedom to enforce quality invigilation.\n\n5. Preventing impersonation by using LMS Logs: Using IT support, we were able to monitor the number of devices our students used to log on to their LMS page. This was to ensure only one device was used in the LMS Examination page activation. This ensured that there was no ghost writer who could take the examination on the studentâs behalf. Along with student gaze and keyboard activity monitoring system, the process ensured that impersonation was impossible on this online examination platform.\n\nTaking into account all these measures, we further had the confidence in the professionalism imparted in our curriculum that is regarded to be essential by many researchers (Ludmerer, 1999, Jaiprakash Mohanraj, 2020), which are tested in such circumstances.\n\n\nResults and discussion\n\nThe proposed proctored, synchronous, closed book, online examinations for barrier examinations were conducted for Year 2 and Year 5 MBBS students from a Malaysian Medical and Healthcare University. We present the findings from the Year 2 Professional MBBS Examinations using the above proposed system. As indicated in Tables 1 and 2, 52% of the student population were women and within this cohort, 52% were international students. The students were between the age group of 18 to 21 years. The exams were proctored by twenty-six lecturers who were aged between 35 to 70 years old. As most of the Teaching and Learning Activities (TLA) activities were conducted on LMS and by using Google Meet during the online session, both students and lecturers were comfortable and welcoming of the use of this new system. All the students took their examinations from the comfort of their homes or other preferred locations. It is worth noting that all the international students (from over eight countries) participated in this examination from their home countries. The full raw data can be found under Underlying data (Mohanraj, 2022).\n\nTest for reliability: Table 3 indicates the mean score of the cohort in all their continuous assessment exams that were conducted face to face and the mean score for the Professional exam conducted online. A test for reliability shown in Table 4 indicates a Cronbach alpha score of 0.714 with indicates that the studentsâ performance across all these exams were reliable and consistent. The results indicate that studentsâ performance in this online examination is consistent with all their previous continuous assessments held earlier in their academic year.\n\nTest for validity of method adopted to conduct online examination: To test the extent to which this assessment tool measures what it claims to measure, we used the following tests.\n\nFace validity: Students and staff perception were taken as feedback after the exam. The results indicate that both staff and students were quite satisfied with the process, its convenience and showed a significant amount of confidence in this system. Table 5 summarizes the studentsâ opinions regarding these exams.\n\nContent validity: Since all the measuring tools used in the face-to-face exams were used in this online exam, none of the assessment strategies from any of the previous examination were altered or modified. This exam tested the delivered curriculum in depth and range which was supported by positive reviews by independent external examiners.\n\nCriterion validity: We tested for correlation between the continuous assessment and the online Professional exam results obtained by this cohort of student. The results indicated in Table 6 shows that, except for the GIN block results, all the other block performance were positively correlated with a significance at the level of 0.01. These results indicate that this online examination method yielded results that reflect the same construct of studentâs performance in continuous assessment. Figure 5 also indicates that the performance of this cohort of students, in terms of pass percentage, was within the range of previous performance observed on the last decade.\n\n** Correlation is significant at the 0.01 level (2-tailed).\n\n* Correlation is significant at the 0.05 level (2-tailed).\n\nThe proctored, synchronous, closed-book, online test described above has been demonstrated to be a trustworthy and legitimate procedure that may be utilised to administer any high-stake exams. With a major portion of 2020 and 2021 affected by COVID-19, medical education has been compelled to shift to the online delivery of content and thus arose the need to find a reliable online method of assessment of the curriculum. Currently, there are varying practices in many schools and universities that have adapted online assessment tools to fit their purpose; however, there are no standard guidelines, reliable tools or methods that have been proven to be reliable alternatives in the education sector. Thus, in this paper, we demonstrated that our suggested examination procedure is an effective instrument for assessing the curriculum and is comparable in reliability, efficiency and convenience of the time-honoured face-to-face mode of assessment. Additionally, we propose that this examination method be utilised in conjunction with assignments to assess students' performance in various distance learning modules.\n\nOne of the most advantageous features of our suggested online testing approach is its low cost. We utilised the current Learning Management System (LMS), which is a free Moodle-based system, in conjunction with the complimentary Google Meet account. The majority of institutions of higher learning have implemented a curriculum delivery method that is heavily reliant on an IT-based support system. This translates into the usage of LMSs for sharing learning materials and administering exams, as well as the use of computer laboratories for administering Computer-Assisted Learning. We have proven that our suggested approach may be used to administer a successful online test utilising the existing technology and essentially at no additional expense.\n\nOur online, proctored examination was very well received by both teachers and students, with particular emphasis on the simplicity with which this innovative approach could be adapted. The student, being tech-savvy, had no trouble understanding the information and following the protocol. Digital divide has been observed between students and lecturers by many researchers (Mohanraj et al., 2019, Sabqat and Khan, 2019), and this seems to be growing with time. In this scenario, our success was with the fact that even with a fair number of the examiners involved there were digital novice and digital immigrants, there were no issues in grasping this technology dependent process. One factor that facilitated the universal acceptability is that there was no requirement to learn a new software, as we used familiar tools and tweaked it to our purpose. Researchers have shown students are subjected to stress in their academic journey and examination was one of the major contributing factors (Sohail, 2013, Fares et al., 2016) that could determine their performance. Thus, our effort was to provide a system that does not add any additional stress on the studentâs process of taking their exams, which was achieved by adapting well known and familiar platforms integrated into the system.\n\nThe limitations in our systems included the necessity to achieve a good student to staff ratio. In a traditional face-to-face environment, the common practice is to assign one staff member to every 20 students; however, in our system, we used one staff member for every six students. We understand that, the lower the staff and student ratio, the better the quality of invigilation. This could be mitigated by engaging part-time invigilator who could be specifically trained in this exercise. Secondly, the strength of the internet connectivity for both the students and invigilators is the backbone of conducting a smooth online examination. This could be hampered due to unforeseen issues or lack of student preparation (with regard to the data package availability). Lastly, any technology dependent system is as good as itâs infra structure and the support staff that maintain it; we recognise that this can be a short coming is some educational institutions. However, since we propose to use only the existing system, a good internet connectivity in the institution with an up-to-date operating system is all that is required to conduct this examination. We recommend that this examination system to be implemented among multiple cohorts both with a university and across different university to improve its reliability and validity.\n\n\nConclusions\n\nThe suggested proctored, synchronous, closed book, online examination can assist in the transformation of manual ways of performing ongoing assessments and high-stake examinations on this online platform for remote learners, which is especially important in these trying times. This system has a high reliability index and repeatability, guaranteeing that the standard and quality of a face-to-face examination are fulfilled in every way.\n\nThis method is extremely cost effective because it does not necessitate the purchase of any additional hardware or software. This form of online examination may be performed at any educational institution with their existing IT support, good internet access, and a resourceful IT support team. Furthermore, because no new software or hardware is utilised, no substantial training/workshop is necessary for invigilators or students to comprehend and operate this method.\n\nThus, we suggest that this proctored, synchronous, closed book, online examination system is reliable, efficient, effective, economical and convenient and recommend it to be used in all the educational sectors that engage in student learning.\n\n\nData availability\n\nFigshare: Data for Repository.xlsx. https://doi.org/10.6084/m9.figshare.20024183.v2 (Mohanraj, 2022).\n\nThis project contains the following underlying data:\n\n- Data for Repository.xlsx [The information provided contains raw student data obtained from multiple summative examination and from different cohorts. All the data provided have been anonymised as per the criteria.]\n\n- FigShare_Online Exam Feedback Form - Student (Responses)-Raw.xlsx\n\nThis project contains the following extended data:\n\n- Student Online Exam Feedback Form.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthor contributions\n\n\n\n⢠The corresponding author is responsible for ensuring that the descriptions are accurate and agreed by all authors.\n\n⢠All the authors were involved in conceptualization and validation of this study.\n\n⢠Jaiprakash Mohanraj (JPM), Ivan Rolland Karkada (IRK) were involved in data collection & resources management.\n\n⢠JPH, Rusli Bin Nordin (RN) & IRK were involved in writing the original draft, review and editing process.\n\n⢠JPM & IRK were involved in Project administration, visualization and writing of the original draft.\n\n⢠JPM, RN & AFS were also involved supervision.",
"appendix": "Acknowledgements\n\nWe would like to express my deepest appreciation to the management of MAHSA University in providing us access to the examination and other necessary support that helped us to complete this research work.\n\n\nReferences\n\nBransford J, Bransford JD, Brown AL, et al.: How people learn: Brain, mind, experience, and school. National Academies Press;1999.\n\nFares J, Al Tabosh H, Saadeddin Z, et al.: Stress, burnout and coping strategies in preclinical medical students. N. Am. J. Med. Sci. 2016; 8: 75â81. PubMed Abstract | Publisher Full Text\n\nJaiprakash Mohanraj HJ: Professionalism in Medical Education: Constructing, Delivering, Assessing & Researching a Framework from a Malaysian Perspective. Int. J. Adv. Sci. Technol. 2020; 29: 13404â13413.\n\nLudmerer KM: Instilling professionalism in medical education. JAMA. 1999; 282: 881â882. PubMed Abstract | Publisher Full Text\n\nMohanraj J: EVIDENCE-BASED ONLINE MODEL PROCTORED EXAMINATION. figshare. Dataset.2022. Publisher Full Text\n\nMohanraj J, Yuen KO, Eshvary P, et al.: Exploring the Digital Divide between Pre-clinical Teachers and Students in an Integrated Medical Curriculum from a Malaysian Private University. Univ. J. Educ. Res. 2019; 7: 1â9. Publisher Full Text\n\nMuzaffar AW, Tahir M, Anwar MW, et al.:2020. A Systematic Review on Online Exams Solutions in E-learning--Techniques, Tools, and Global Adoption. arXiv preprint arXiv:2010.07086.\n\nPreston R, Gratani M, Owens K, et al.: Exploring the impact of assessment on medical studentsâ learning. Assess. Eval. High. Educ. 2020; 45: 109â124. Publisher Full Text\n\nReyna J: Twelve Tips for COVID-19 friendly learning design in medical education. MedEdPublish. 2020; 9. Publisher Full Text\n\nSabqat M, Khan RA: Exploring the digital divide between medical students and medical teachers. Health Prof. Educ. J. 2019; 2: 39â46. Publisher Full Text\n\nSohail N: Stress and academic performance among medical students. J. Coll. Physicians Surg. Pak. 2013; 23: 67â71. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "290476",
"date": "03 Jul 2024",
"name": "Aisha Rafi",
"expertise": [
"Reviewer Expertise Medical education",
"Anatomy"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study has introduced an online assessment model developed during COVID-19 lockdown. The nine steps modified fish bone online examination model (MFBOEM) has been developed after approval from University senate, faculty board and assessment committee. Clear guidelines and SOPs were established to guide students and invigilators, covering everything from initial communication to technical setup and detailed instructions for practice and final exams. The Learning Management Systems (LMS) and Google Meet were used as examination tools. The validity of the exam was ensured by various measures for online proctoring such as using two cameras for monitoring, invigilators tracking the eye movements and keyboard activities of students, and requiring students to show their surroundings before starting the exam and keep the devices unmute throughout the examination. A real-time, proctored, closed-book mock exam for Year 2 and 5 MBBS was conducted. Â The feedback was collected to refine the process. The online final professional exam was reliable, with a Cronbach alpha score of 0.71. The online exam received positive feedback from both students and faculty, indicating high satisfaction levels. The MFBOEM is cost-effective as it requires no additional hardware or software, making it suitable for institutions with existing technology. The results of the online exams were consistent with traditional face-to-face exams, demonstrating the model's effectiveness. The authors recommend this method as a reliable, efficient, and economical alternative to traditional exams for educational institutions, especially during times when in-person exams are not feasible. The review suggests a few corrections. The abstract mentions the year 2 students were examined only whereas in the methodology section it was stated year 5 and year 2 final professional examinations were conducted online. The past and future tense is used while describing the procedure. Similarly past and present tense were used in the feedback form. Use past tense only. Table 3 does not mention the professional exam mean score conducted online. It only states the CA scores. Face validity is not clear. Criterion validity states last decade, please correct it.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "290474",
"date": "19 Jul 2024",
"name": "Maggie Hartnett",
"expertise": [
"Reviewer Expertise Digital education",
"higher education",
"online proctored exams"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to read the manuscript of a solution adopted by one university to replace f2f invigilated exams with online, synchronous, invigilated exams.\nWhile I have answered yes to the above questions (because the paper does meet those criteria) this is not what I would consider a research paper nor a methods paper. It is a descriptive account of a set of procedures that were put in place to conducts OPEs (online proctored exams) during the pandemic. While interesting to some degree, it does not take into account any of the existing OPE research and what we know and understand about OPEs especially since the pandemic, which is considerable.\nThe results provided don't add a great deal to the research field apart from a detailed account of what they did that someone else could follow if they chose to. The authors argued that one of the strengths of the approach was the lack of additional expense but they neglected to include the significant amount of time required by a range of people to establish the approach and the need for significantly more invigilators than in f2f situations.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-249
|
https://f1000research.com/articles/12-248/v1
|
07 Mar 23
|
{
"type": "Case Report",
"title": "Case Report: A Rare Case of Eosinophilic Ureteritis in a Woman with Reccuring Bilateral Ureteric Strictures causing Hydroureteronephrosis",
"authors": [
"Sahat Basana Romanti Ezer Matondang",
"Adistra Imam",
"Guntur Darmawan",
"Armand Achmadsyah",
"Ibrahim Abubakar Hilmy",
"Andrian Harsanto",
"Rochani Sumardi",
"Adistra Imam",
"Guntur Darmawan",
"Armand Achmadsyah",
"Ibrahim Abubakar Hilmy",
"Andrian Harsanto",
"Rochani Sumardi"
],
"abstract": "Introduction: Eosinophilic ureteritis is a very rare etiology of ureteric obstruction that leads to hydroureteronephrosis. We have only so far discovered a few case reports discussing eosinophilic ureteritis and the exact cause of this condition is poorly understood. Clinical findings: we report a very interesting case of a 71-year-old patient presenting with general weakness and oliguria, computed tomography (CT) scan found bilateral hydroureteronephrosis caused by recurring ureteric strictures. Diagnosis, interventions, and outcomes: ureterorenoscopy (URS) to extract a specimen for biopsy and laser fulguration to relieve ureteral strictures was performed. Double-J (DJ) stents were inserted both in the left and right ureters to overcome the ureteric strictures. Microscopy of the excised specimen revealed dominant eosinophilic infiltration. Therefore, diagnosis of eosinophilic ureteritis was made. In addition to stenting, Meropenem and Mirabegron was given to the patient. After a week of observation, the patient made a full recovery with no signs of initial symptoms. Conclusion: It is important to consider eosinophilic ureteritis in the differential diagnosis of any ureteric stricture because it is a highly uncommon entity with variable clinical characteristics. The gold standard procedure, which should be utilized to make the diagnosis, is a histological examination.",
"keywords": [
"Eaosinophilic ureteritis",
"ureteric stricture",
"hydronephrosis",
"ureteroscopy"
],
"content": "Introduction\n\nThe term âhydroureteronephrosisâ refers to a condition in which the renal pelvis, calyces, and ureter enlarge due to restriction of the free flow of urine from the kidney, which causes the renal cortex to gradually atrophy.1 It can be caused by a structural or physiological process that prevents urine from flowing normally.1 This can occur anywhere from the kidneys to the urethral meatus.1 The etiology, location, and duration of the obstruction may affect the symptoms. If the etiology is ureteral calculus and occurs suddenly, the symptoms often present with colicky pain in the flanks.1 The-non calculus etiology of hydroureteronephrosis differs depending on the patientâs age and gender.1 The most frequent cause in children is congenital obstruction of the pelvi-ureteric junction (PUJ) and posterior urethral valves, whereas the most frequent causes in adult males are benign prostatic hypertrophy (BPH), urethral/ureteric strictures, bladder cancer, and obstruction of the bladder outlet.1 It also happens secondary to pregnancy in childbearing females, but in older people, the major causes are cervix and ovary carcinomas.1\n\nEosinophilic ureteritis is a very rare etiology of ureteric obstruction that leads to hydroureteronephrosis, and to date the diagnosis of these findings can only be made through pathologic examination.2 We have only so far discovered a few case reports discussing eosinophilic ureteritis and the exact cause of this condition is poorly understood, but there may be some association with atopy, hypereosinophilic syndrome, and prior ureteral interventions.2 Here, we report a very interesting case of a 71-year-old patient presenting with bilateral hydroureteronephrosis caused by recurring ureteric strictures of eosinophilic ureteritis etiology.\n\n\nCase report\n\nA 71-year-old Indonesian housewife presented to our private hospital located in Centre of Jakarta, Indonesia with complaints of general weakness and oliguria. No previous episodes of fever, hematuria, dysuria, or flank pain were noted. The patient had already been diagnosed with bilateral ureteric stenosis six years ago and has had a double-J (DJ) stent for about four months. Since then, the patient had a record of recurrent urinary tract infections with only fever and incomplete emptying when urinating as symptoms. The patient had a history of lung tuberculosis 50 years ago that was treated until they recovered completely, unintentional weight loss of more than 10 kilograms last year, multinodular non-toxic goiter treated once daily with thiamazole 10 mg, and right breast tumour that has not been further diagnosed. There was no relevant family and psycho-social history that can cause the current symptoms. The physical examination, upon admission, revealed both conjunctival were pallor and no tenderness in both renal angles. According to a urine analysis that we collect from a mid-stream urine put in a sterile cup, there were packed pus cells per High Power Field (PHF), 1 red blood cell/HPF, 1+ protein, 1+ bacteria, and 1+ leukocyte esterase. The patientâs baseline investigations, upon admission, revealed red blood cell count was 6.3 g/dL (normal range 12.0-14.0 g/dL) with mean corpuscular volume (MCV) 85.8 fl (normal range 82.0-92.0), mean corpuscular haemoglobin (MCH) 29.4 pg (normal range 27.0-31.0), mean corpuscular haemoglobin concentration (MCHC) 34.3% (normal range 11.5-14.5%), white blood cell count was 6560/mm3 (normal range 5000-10000/mm3), with 3% eosinophils (normal range 1-3%). Thrombocytes was 361000/ÎŒL (normal range 150000-400000/ÎŒL). Thyroid stimulating hormone (TSH) was 1.8270 ÎŒIU/mL (0.3500-4.9400 ÎŒIU/mL) and Free T4 (FT4) was 0.79 ng/dL (0.70-1.48 ng/dL) which indicated the pateintâs thyroid level was within normal limit. A renal function test showed urea was 58 mg/dL (normal range 14-38 mg/dL) and serum creatinine was 3.39 mg/dL (normal range 0.55-1.02 mg/dL). The urine culture sample was obtained from the catheter using surface streak method. It showed escherichia coli infection with >100000 Colony-forming unit (CFU) (normal range <10000 CFU). Gene-Xpert (sample taken from patientâs sputum during hospitalization) and Interferon-gamma release assay (IGRA) (sample taken from blood obtained during hospitalization) showed negative signs of tuberculosis infection. Galactomannan test (sample taken from blood obtained during hospitalization) also revealed negative sign of aspergillosis infection.\n\nComputerized tomography (CT) urogram without contrast was performed to investigate further the cause of patientâs complaints as lab results doesnât point to a specific diagnosis.23 CT Urogram revealed the thickening of the right proximal ureteric wall up to the right mid-ureteric wall (Figure 1A) and thickening of the left proximal ureteric wall (Figure 1B) suggestive of ureteric stricture with severe bilateral hydronephrosis (Figure 1A,B). There was no diagnostic challenge during hospitalization. A pre-operative working diagnosis of bilateral ureteric stricture and Grade 4 hydronephrosis was made. Before the patient underwent surgery, 750 cc of packed red blood cells were transfused until the patientâs red blood cell count was 10.8 g/dL (normal range 12.0-14.0 g/dL). Then, bilateral diagnostic ureterorenoscopy (URS) under spinal anesthesia was performed with semi-rigid ureteroscope. Urine cytology revealed milky urine in the urinary tract that contains lots of neutrophils, lymphocytes, macrophages, and urothelial cells the (Figure 2), no malignant cells was found. Tuberculosis-polymerase chain reaction (TB-PCR) of urine showed no trace of tuberculosis. Retrograde Pyelography (RPG) showed multiple bilateral ureteral strictures (Figure 3) and then the scope was negotiated up to the right-left ureter to confirm our findings and tissue samples were obtained for biopsy (Figure 4). Subsequently, laser fulguration was done to release ureteral strictures from both ureters (Figure 5). Microscopy of the excised specimen revealed dominant eosinophilic infiltration along with atypical stromal cells (Figure 6). After that, a DJ stent was successfully inserted both in the left and right ureters (Figure 7). A diagnosis of eosinophilic ureteritis was made based on this pathological finding. We decided to give the patient iv meropenem 3Ã1 gram for seven days during hospitalization and oral mirabegron 1Ã50 mg to overcome patientâs overactive bladder. After seven days, the patient made a complete recovery with no sign of general weakness, oliguria, and urinary tract infection from the patientâs second urine analysis. We followed-up the patientâs condition for another week and discovered there were no sign of overactive bladder and oliguria.\n\n(A) Coronal view, thickening of the right proximal ureteric wall up to right mid ureteric wall (white arrow); (B) Coronal view, thickening of the left proximal ureteric wall (white arrow).\n\nNo sign of malignancy cell with lots of neutrophil, lymphocyte, macrophage, and urothel.\n\n(A) Visible Milky Urine in the bladder and ureter; (B) Mass that causing stricture in ureter bilateral.\n\n(A) Laser fulguration in left ureter for biopsy; (B) Laser fulguration in right ureter for biopsy.\n\nWall of the ureter containing a dense mural infiltrate composed predominantly of eosinophils.\n\n\nDiscussion\n\nEosinophil is one of blood granulocytes derived from bone marrow. Production of eosinophil required a complex interaction between IL-5, IL-3, and GM-CSF, which were mediated by T-helper 2 cellsâ immune response. Eosinophil acts by secreting proteolytic enzymes which result in cell wall damage of targeted pathogens, mostly parasites.3,4 Eosinophils may reside in several tissues, mostly located in the respiratory and gastrointestinal tract. The presence of eosinophils in the urinary tract is deemed pathologic, such as bladder and ureter.5\n\nEosinophilic ureteritis is a very rare clinicopathological entity known to cause ureteric stricture.6 The symptoms may vary, although most of the patients reported the symptoms mimicking urinary stones with or without urinary tract infection (UTI). In our patients, the main symptoms were recurring UTIs despite no history of colicky pain suggesting urinary stone.7\n\nTo our knowledge, the present case is the second reported example of eosinophilic ureteritis causing bilateral upper ureteric strictures and bilateral hydronephrosis in adults. The first reported case was from Singh et al.7 who also reported bilateral upper ureteric strictures with hydroureteronephrosis.7 Another study conducted by Bajracharya et al.2 reported a case of unilateral ureterohydronephrosis also related to eosinophilic ureteritis.2\n\nThe exact etiology of the disease remains unclear. In our patient, we previously suspected a tuberculous infection as 50 years ago the patient was infected with this disease. However, there was no evidence of tuberculous infection in the ureter from the patientâs urine cytology, GeneXpert and IGRA tests were also negative. Previous studies reported the possibility of prior parasites infection such as Schistosoma, Toxocara, and Sparganum in the bladder as the disease etiology. Filarial infection has also been reported as a possible trigger of the condition.7 It is hypothesized that in parasite infections, activated eosinophils released cytotoxic cationic proteins which target surrounding tissues, further resulting in chronic inflammation with scattered eosinophils that lead to fibrous formation.8 Another possible pathophysiology is IgE-mediated release of eosinophilotrophic molecules.9 Presence of other organisms (Enterobacter aerogenes or fungal Candida albicans) has also been reported in other organs, such as eosinophilic cholangitis. In our case, the patientâs galactomannan test revealed to be negative and we didnât find any stigmata of filariasis.10\n\nOur patient is currently treated for multinodular non-toxic goiter with thiamazole. Hyperthyroidism has also been associated with peripheral eosinophilia. The link between hyperthyroidism with eosinophil-induced organ damage has not been clearly reported, although relative cortisol deficiency in thyrotoxicosis has been associated with high eosinophil count.11 Another possible connection in our patient was related to the breast tumour, in which 3.7% of patients with breast tumour has been associated with tissue eosinophilia.12 Although, further studies are needed to evaluate these possible etiologies of eosinophilic ureteritis due to lack of evidence.\n\nStudies regarding eosinophilic ureteritis are limited, although eosinophil-induced inflammation in other genitourinary organs such as the bladder has been reported.13 Many factors have also been associated with eosinophilic inflammation, including previous medication (such as warfarin, methicillin, intravesical mitomycin, thiotepa, and anthranilic acid), atopy (food or other allergens) or autoimmune diseases, previous operative procedure, and trauma. In our case, the patient never had any previous history of the above-mentioned medications.13,14\n\nEosinophil-induced organ damage has also been reported to be linked to one another, in line with a study by Kim et al.15 who showed an association between eosinophilic cystitis and enterocolitis.15 Similar pattern has also been shown by Platt et al.16 who showed an association between eosinophilic ureteritis and eosinophilic cholangitis.16 Eosinophilic ureteritis has also been associated with hypereosinophilic syndrome, marked by persistent blood eosinophilia (>1.5 Ã 109/L in six or more consecutive months) with eosinophil-induced organ damage without evidence of active allergic and parasitic causes, or malignant disorders.17 Examples of eosinophil-induced organ damage are eosinophilic gastroenteritis, esophagitis, dermatitis, pneumonia, and fasciitis. The etiology of this syndrome was also unknown, but cryptic cytogenetic abnormality in eosinophils has been identified. Since we couldnât find any etiology linked to eosinophilic ureteritis in our patient, the main cause remained idiopathic. 17,18\n\nRegarding the disease rarity, care needs to be taken into account while encountering patients with recurrent UTIs without marked laboratory parameters. Before eosinophilic ureteritis was suspected, prior exclusion to active parasitic infection, genitourinary malignancies, and calculus are needed. In eosinophilic ureteritis, the main diagnostic tool is a histopathological examination using biopsy.2,6,7,19\n\nRadiologically, the patient presented a nonspecific pathology which shows ureteral wall thickening suggestive of stricture that can be found in eosinophilic ureteritis, 7 but these findings can also be found in other inflammatory or neoplastic cases such as in genitourinary tuberculosis or carcinoma of the urinary tract.20,21 As such, a radiological diagnosis is unclear and there is a need for pathological examination to diagnose the specific cause of the disease and guide the clinician to the right therapy.\n\nIn our patient, after confirmation of tissue eosinophilia suggesting the presence of eosinophilic ureteritis, double-J (DJ) stent was inserted to overcome the ureteric stricture. There has not been any accepted consensus regarding the diseaseâs management, although the use of oral corticosteroids (e.g., prednisolone) and non-steroidal anti-inflammatory drugs (e.g., diclofenac) has been reported as the initial conservative therapy with subsequent ureteral obstruction remission. Other oral drugs include antihistamines and antibiotics. Cyclosporine and azathioprine can be given in refractory cases. We decided to administer meropenem as an antibiotic as the patient already had recurrent urinary tract infections and a history of prolonged usage of antibiotics 13,19\n\nMore invasive treatment may be needed in patients with evident hydronephrosis. DJ stenting may be performed to evacuate the obstruction related to ureteral stricture. However, the insertion of stent or catheterization procedure has been paradoxically associated with a case of eosinophilic ureteritis in already present eosinophilic cystitis. This may also lead to potential recurrence due to persistent inflammatory reactions after instrument insertion in the genitourinary tract.22\n\nRegarding the above-mentioned risks, surgical treatment has been shown superior in the management of eosinophilic ureteritis.13 Previous studies have performed variable surgical procedures, ranging from total nephroureterectomy to ureteral segmentectomy in the stenotic segment with subsequent end-to-end anastomosis. After this treatment, the patient refused further surgery after some considerations because they wanted to observe how the current treatment goes first. The patient is also scheduled for another URS evaluation in the next six months, then whether reconstructive surgery or conservative treatments using DJ stents are needed will be determined afterward. Routine monitoring should be done to evaluate the patientâs clinical signs and symptoms.2\n\nThis case report is limited by the short follow-up period and lack of information from patientâs past medical history as she was admitted not in our hospital. We also couldnât find any literature discussing relationship between eosinophilic ureteritis and gender. However, its strength is that as we directly observed the patient rather than taking information retrospectively, this may reduce any potential bias. We also have excluded most of the potential etiology for ureteric strictures.\n\nIn conclusion, it is important to consider eosinophilic ureteritis in the differential diagnosis of any ureteric stricture because it is a highly uncommon entity with variable clinical characteristics. The gold standard procedure, which should be utilized to make the diagnosis, is a histological examination. For improved results, early detection, and prompt treatment are expected.\n\nWritten informed consent for publication was taken from the patient. Authorizing the use of their clinical information to be used in this case report.",
"appendix": "Data availability\n\nZeonodo: A Rare Case of Eosinophilic Ureteritis in a Woman with Reccuring Bilateral Ureteric Strictures causing Hydroureteronephrosis: Supplemental CT Scan Data, https://doi.org/10.5281/zenodo.7622523. 23\n\nThis project contains the following extended data:\n\n- All DICOM CT images.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nIqbal S, Raiz I, Faiz I: Bilateral hydroureteronephrosis with a hypertrophied, trabeculated urinary bladder. Malaysian J. Med. Sci. 2017; 24(2): 106â115. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBajracharya UB, Sah AK, Baral H, et al.: Eosinophilic Ureteritis Causing Ureterohydronephrosis: A Case Report. Med. J. Shree Birendra Hosp. 2021; 20(2): 170â172. Publisher Full Text\n\nKovalszki A, Weller PF: Eosinophilia. Prim. Care Clin. Off. Pract. 2016 Dec; 43(4): 607â617. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiao W, Long H, Chang CCC, et al.: The eosinophil in health and disease: From bench to bedside and back. Clin. Rev. Allergy Immunol. 2016; 50(2): 125â139. PubMed Abstract | Publisher Full Text\n\nMosholt KSS, Dahl C, Azawi NH: Eosinophilic cystitis: Three cases, and a review over 10 years 1935. BMJ Case Rep. 2014; 2014: bcr2014205708. Publisher Full Text\n\nSpark RP, Gleason DM, DeBenedetti CD, et al.: Is eosinophilic ureteritis an entity? 2 case reports and review. J. Urol. 1991; 145(6): 1256â1260. PubMed Abstract | Publisher Full Text\n\nSingh JC, Karnik SV, Gopalakrishnan G: Eosinophilic ureteritis presenting as bilateral upper ureteric strictures. Scand. J. Urol. Nephrol. 2004; 38(3): 260â262. PubMed Abstract | Publisher Full Text\n\nHellstrom HR, Davis BK, Shonnard JW, et al.: Eosinophilic pyeloureteritis: Report of a case. J. Urol. 1979; 122(6): 833â834. PubMed Abstract | Publisher Full Text\n\nDubucquoi S, Janin A, Desreumaux P, et al.: Evidence for Eosinophil Activation in Eosinophilie Cystitis. Eur. Urol. 1994; 25(3): 254â258. Publisher Full Text\n\nRodgers MS, Allen JP, Koea JB, et al.: Eosinophilic cholangitis: a case of âmalignant masqueradeâ. HPB. 2001 Sep; 3(3): 235â239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim MJ, Yoo KH, Kim BK, et al.: Two Cases of Apathetic Hyperthyroidism associated with Peripheral Eosinophilia. J. Korean Soc. Endocrinol. 2005; 20(1): 78. Publisher Full Text\n\nChouliaras K, Tokumaru Y, Asaoka M, et al.: Prevalence and clinical relevance of tumor-associated tissue eosinophilia (TATE) in breast cancer. Surgery. 2021 May; 169(5): 1234â1239. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nSergeant G, Slabbaert K, Werbrouck P: Recurrent flank pain caused by eosinophilic ureteritis mimicking urinary stone disease: A case report. Int. Urol. Nephrol. 2003; 36(1): 23â25.\n\nStormont G, Makari J, Bedrnicek JB, et al.: Eosinophilic Ureteritis in a Child With Ureteropelvic Junction Obstruction and Vesicoureteral Reflux. Cureus. 2020; 12(7): 1â9. Publisher Full Text\n\nKim MS, Park H, Park CS, et al.: Eosinophilic cystitis associated with eosinophilic enterocolitis: case reports and review of the literature. Br. J. Radiol. 2010 Jun; 83(990): e122âe125. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPlatt ML, Kiesling VJ, Vaccaro JA: Eosinophilic ureteritis associated with eosinophilic cholangitis: A case report. J. Urol. 1990; 144(1): 127â129. PubMed Abstract | Publisher Full Text\n\nHarris A, Sc M, Johnson DW, et al.: A Tyrosine Kinase Created by Fusion of the PDGFRA and FIP1L1 Genes as a Therapeutic Target of Imatinib in Idiopathic Hypereosinophilic Syndrome. N. Engl. J. Med. 2003; 348(13): 1201.\n\nRoufosse FE, Goldman M, Cogan E: Hypereosinophilic syndromes. Orphanet J. Rare Dis. 2007 Dec 11; 2(1): 37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nManimaran D, Karthikeyan TM, Sreenivasulu M, et al.: Eosinophilic cystitis mimicking bladder tumour - A rare case report. J. Clin. Diagn. Res. 2013; 7(10): 2282â2283.\n\nJung YY, Kim JK, Cho KS: Genitourinary Tuberculosis: Comprehensive Cross-Sectional Imaging. Am. J. Roentgenol. 2005 Jan 1; 184(1): 143â150. PubMed Abstract | Publisher Full Text\n\nVikram R, Sandler CM, Ng CS: Imaging and Staging of Transitional Cell Carcinoma: Part 2, Upper Urinary Tract. Am. J. Roentgenol. 2009 Jun 1; 192(6): 1488â1493. PubMed Abstract | Publisher Full Text\n\nMitas JA, Thompson T: Ureteral involvement complicating eosinophilic cystitis. Urology. 1985; 26(1): 67â70. PubMed Abstract | Publisher Full Text\n\nMatondang SBRE: A Rare Case of Eosinophilic Ureteritis in a Woman with Reccuring Bilateral Ureteric Strictures causing Hydroureteronephrosis: Supplemental CT Scan Data. [Data]. Zenodo. 2023. Publisher Full Text"
}
|
[
{
"id": "168181",
"date": "13 Apr 2023",
"name": "Su-Xia Wang",
"expertise": [
"Reviewer Expertise Pathology",
"renal disease",
"immunology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report described an usual eosinophilic ureteritis as the causes of ureteric obstruction leading to hydroureteronephrosis, which provides an important clue in the differential diagnosis of ureteric stricture.\nThere are some points to be addressed:\nAlthough a relatively complete examination has been performed to exclude the causes of eosinophilic ureteritis, the indolent infection of any possible organisms should be scanned by new technique, such as NGS for micro-organisms.\n\nThe infiltration of eosinophils in the urinary tract suggested the cytokines induced pathophysiology processes. It is suggested to measure the level of cytokines in blood, including IL-6, TNF-a, etc.\n\nHowever, the possible etiology of this patient in unclear. The recurrent E. coli infection of urinary tract may be the triggers of eosinophilic inflammation, it should be discussed in detail.\n\nThe body of manuscript is too long and fussy, it is suggested to write in a concisely and refined style.\n\nIs the background of the caseâs history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-248
|
https://f1000research.com/articles/12-138/v1
|
06 Feb 23
|
{
"type": "Review",
"title": "Self-empowerment of life through RNA networks, cells and viruses",
"authors": [
"Luis Villarreal",
"Guenther Witzany",
"Luis Villarreal"
],
"abstract": "Our understanding of the key players in evolution and of the development of all organisms in all domains of life has been aided by current knowledge about RNA stem-loop groups, their proposed interaction motifs in an early RNA world and their regulative roles in all steps and substeps of nearly all cellular processes, such as replication, transcription, translation, repair, immunity and epigenetic marking. Cooperative evolution was enabled by promiscuous interactions between single-stranded regions in the loops of naturally forming stem-loop structures in RNAs. It was also shown that cooperative RNA stem-loops outcompete selfish ones and provide foundational self-constructive groups (ribosome, editosome, spliceosome, etc.). Self-empowerment from abiotic matter to biological behavior does not just occur at the beginning of biological evolution; it is also essential for all levels of socially interacting RNAs, cells and viruses.",
"keywords": [
"RNA stem-loop groups",
"social behavior",
"biocommunication",
"gangs of life",
"memory",
"learning",
"excess productivity"
],
"content": "1. Introduction\n\nIn the 20th century, when biology was a subdiscipline of physics and chemistry, it was common to choose explanatory models that helped us understand evolutionary processes in terms of gradual steps from abiotic physical reactions to biological variation and selection processes (Eigen, 1971; Schuster, 2011). With the rise of quasispecies theory and research into the behavior of RNA viruses, we now know that RNA group interaction motifs âresemble, in many ways, social behaviorâ (see Eigen, 1971, p. 505). We can use a more up-to-date description that integrates an abundance of signal-mediated interactions into a more coherent picture. These interactions range from social RNA networks and cellâcell communication, to communication of viruses (Villarreal & Witzany, 2010, 2013a, 2015; Witzany, 2015). This picture contains features that are not exclusively the domain of mathematics, physics and chemistry but also come under empirical social sciences that help us better understand various behavioral motifs in group interactions (DÃaz-Muñoz, Sanjuán, & West, 2017; Witzany, 2016a).\n\n\n2. At the evolutionary beginning of biotic behavior\n\nSuccessful concepts about prebiotic evolution and its various implications have been reviewed elsewhere and will not be repeated here (Oparin, 1965; Oparin & Gladilin, 1980; Mathis et al., 2017; Vlassov et al., 2004; Lehman, 2013). We are more interested in the real beginnings of biological evolution. Before the start of biotic processes, we can identify a kind of self-organization of matter without biotic features: the snap-back of single-stranded RNA molecules. This mechanism is found when a few single-stranded RNA molecules fold back within their own identity, to form a double-stranded RNA stem and a single-stranded RNA loop at the folding angle (Manrubia & Briones, 2007; Mattick & Amaral, 2022; Stich, Briones, & Manrubia, 2007; Levin, Gandon & West, 2020). This results in the basic structure of all subsequent following RNA stem-loop group interaction motifs (Moore, 1999; MÃŒller et al., 2012; Petkovic & MÃŒller, 2013). The double-stranded RNA stem is not prone to binding based on the complementarity of RNA syntax. In contrast, the single-stranded RNA loop is somewhat prone to binding as it complementarily binds to other single-stranded RNAs. The snap-back functions exclusively according to the laws of physics and chemistry. No biological selection occurs at this stage. It clearly represents self-organization of matter.\n\nThe RNA stem-loops have several distinct parts/subunits: stems consisting of base-paired nucleotides and loops/bulges/junctions consisting of unpaired regions limited by stems. It is important to note that any RNA is part of such stem-loops. Biological selection processes emerge in the presence of a certain density of RNA stem-loops. Then RNA stem-loops cooperate and compete, which are behavioral motifs that are completely absent in abiotic matter (see Figure 1). This means that biological selection is clearly a result of social interactions and starts not with the first living cell or with LUCA (last universal common ancestor), but earlier in the RNA world. An unexpected finding is that cooperative RNA stem-loops outcompete selfish ones (Smit, Yarus & Knight, 2006; Hayden & Lehman, 2006; Higgs & Lehman, 2014; Vaidya et al., 2012; Vaidya, Walker & Lehman, 2013; Witzany, 2016b).\n\nAccording to Villarreal, group identity and cooperativity of an RNA collective require opposing functions which are essential for the genesis of life (social behavior of agents). Interestingly, at the very beginning of the RNA world, ribozyme consortia with group identities initiated the differentiation competence of âselfâ from ânon-selfâ, like âgangsâ, according to dynamically varying contextual requirements. Figure 1 assembles all key topics: abioticâbiotic interactions, consortial life and identity building, inclusionâpreclusion, opposing ribozyme activities (endonucleaseâligase), basics of cell biology and code emergence out of interactions (from Villarreal, 2015; with permission by the New York Academy of Sciences).\n\nThe abundance of RNA agents that form cooperative groups seem to have their first evolutionary revolution in forming the tRNA clover loop with three stem-loop arms, which is a forerunner of the more complex assembly of ribosomal subunits (Wegrzyn & Wegrzyn, 2008; Kanai, 2015). The clover leaf consists of an acceptor stem, D-arm, the anticodon arm, the variable loop, and the T-arm, also known as the TΚC arm. Each arm consists of a double-stranded stem and a single-stranded loop (Root-Bernstein, Kim, Sanjay, & Burton, 2016; Kim et al., 2017; Sun & Caetano-Anolles, 2007; Di Giulio, 2012). tRNAs appear to represent the oldest consortia of RNA stem-loop groups and are derived from two different parts that originally emerged for purposes other than the translation of mRNAs into polyaminoacid sequences (Caetano-Anolles & Sun, 2014; Root-Bernstein & Root-Bernstein, 2019; Demongeot & Seligman, 2019; Dantas, José, & de Farias, 2021). As tRNAs are one of the most prominent RNA groups with a long history, this structure has been conserved for all organisms thanks to the evolutionary, successful and therefore beneficial selection (Schmidt & Matera 2020). It is also interesting to note that the pre-tRNAs contain introns that must be spliced out before a final mature tRNA can start translation processes (Hayne, Lewis & Stanley, 2022).\n\nThe self-ligation of different parts of RNA stem-loop groups occurs at single-stranded RNA parts (Gwiazda et al., 2012). Here, it is important to note that circular RNAs are protected against endonuclease degradations and therefore represent an evolutionary benefit in this early stage of the RNA world (Lasda & Parker, 2014; MÃŒller, 2015; Lee & Koonin, 2022; Rivera-Madrinan, Di Iorio, & Higgs, 2022). With the emergence of the RNA polymerase ribozyme, functional RNA molecules, including the polymerase itself, could be copied (Attwater, Wochner, & Holliger, 2013; Tjhung, Shokhirev, Horning, & Joyce, 2020). Early in the RNA world an important player emerged, RNAse H, which may have evolved from ribozymes, related to viroids and forming ribosomes. Even today, RNAse H mediates a variety of functions in all domains of life, including the virosphere (Moelling, Broecker, Russo, & Sunagawa, 2017). RNA stem-loop groups thus lay the foundations for complex essential â-someâ activities, starting with ribosomes.\n\nWith the emergence of ribosomes, the next biological revolution took place: protein translation. The RNA polymerase has a close interaction with the ribosome, which helps to maintain genome integrity and conserves energy during transcription and translation (McGary & Nudler, 2013). The rather precise translation into proteins paved the way to a wide range of cellular domains (Gordon, 1995; Davidovich, Belousoff, Bashan, & Yonath, 2009; Bernhardt, & Tate, 2010; Sun & Caetano-Anollés, 2008). Interestingly, all known cells share this ribosomal feature, but no virus contains it, although RNA stem-loop groups in the long evolutionary process not only assembled but also genetically fixed some ribosomal subunits (Mizuno et al., 2019; Bowman et al., 2020). Furthermore, the ribosome is a ribozyme that is at the core of the translation into proteins which do the most work in cell biology. It enables the tRNA to build a series of polypeptides of amino acids, which are the basics for all functions of cellular organisms. In addition, the formation of a tri-peptide at the tRNA is a concerted action at the center of this ribosome ribozyme. Meanwhile, the evolutionary history of the ribosomal subunits has been extensively investigated (Randau & Söll, 2008; Harish & Caetano-Anolles, 2012; Petrov et al., 2015). Each of these big consortia of single RNA stem-loops can be studied according to context and history, which means that the various very old parts and the younger parts of the ribosomal subunits can be analyzed (Ariza-Mateos et al., 2019). The question remains as to how and why these two different consortia are unified and conserved in any cellular life.\n\nThe messenger RNA derives from a pre-stage as the primary transcript out of the DNA of a gene performed and produced by an RNA polymerase. Without polymerases, no single DNA strand can be processed into an mRNA. Within the pre-mRNA primary transcript, we can find the protein-coding sequences (non-repetitive) and the introns (repetitive) that separate the final mRNA, ready to be translated into the polypeptide strain. The evolutionary split of RNA sequences into repetitive and non-repetitive nucleotide syntax had far-reaching consequences for the evolution of protein-coding complexity and regulation (Shapiro & von Sternberg, 2005; Jurka et al., 2007; Witzany, 2017a).\n\nVarious highly coordinated processes are outlined by different RNA networks, such as RNA editing (editosome) and alternative splicing (spliceosome). Both the editosome and the spliceosome represent RNA stem-loop groups that assembled through ligation procedures during their long evolutionary history (Gott, 2003; Matlin & Moore, 2007; Alfonzo, 2008; Hesselberth, 2013). This history is illustrated by the variety of, for example, six subunits of the final spliceosome (a group of nuclear RNAs). Spliceosomal actions take place after the RNA editing by the editosome. Splicing and editing are heavily interconnected and provide variable meanings of the identical DNA sequence. For translation into cellular organisms, a line-up of all the exons must be produced in which the introns are cut out. The sites where splicing of the introns occurs must be exactly identified (Matera & Wang, 2014; Izquierdo & Valcárcel, 2006). Interestingly, small nuclear RNAs (snRNA) base-pair with short sequences in pre-mRNAs to mark the sequences to be spliced out. A total of 200 small nucleolar RNAs are known to act as guides (Zhang et al., 2019). They are encoded in introns and transcribed by RNA polymerase II. In some organisms, these introns are conserved more than the exons. Following the splicing processes, the remaining RNAs are recycled for other catalytic processes.\n\nRNA stem-loop groups represent an unmanageable quantity of sophisticated regulatory networks (Mattick & Amaral, 2022). These groups are crucial in the following functions:\n\n⢠DNA replication with important functions of centromeres and telomeres in genome maintenance\n\n⢠RNA guidance of chromosome structure\n\n⢠Regulation of transcriptional and post-transcriptional modifications by spliceosomes and editosomes according to the requirements of the context\n\n⢠Regulatory pathways and coordination in all steps of the translation into proteins\n\n⢠Epigenetic marking and short-term and long-term memory formation and its (re)modification\n\n⢠DNA repair organization and coordination in all detailed steps and substeps\n\n⢠Immunity organization and coordination in all steps and substeps by genome plasticity, V (ariable) D (iversity) J (oining) plasticity in adaptive immune response\n\n⢠Genetic identity of organisms which initiates motifs of self-interaction or non-self-interaction\n\n⢠Genetic content composition of host organisms by genetic parasites (viruses and defectives such as transposons and retroposons)\n\n⢠Intron/exon genome fragmentation as a benefit in immune functions (CRISPR/Cas) as well as in genome modularity and complexity.\n\nThe active roles played by RNA stem-loop groups ensure all life processes currently known and start with the transcription process out of the relatively stable DNA storage medium. After transcription, an abundance of RNA stem-loop variants are available and interact in well-coordinated actions. The folding loop remains as a single-stranded RNA sequence. Various motifs have been identified, yet all of them share a common function: they stabilize RNA tertiary formation. Such motifs include:\n\n⢠pseudoknots, kissing loops, A-minor motifs, A-platforms, kink-turns, S-turns, tetraloops and their receptors, and a variety of non-canonical base-pairs and base-triples\n\n⢠ribosomal frameshift as a natural technique to process alternative translation of an mRNA sequence by changing the open-reading frame\n\n⢠bypassing translation\n\n⢠competing endogenous RNAs.\n\nAll these highly coordinated and interconnected motifs of RNA stem-loop groups may alter the meaning of the information stored in the DNA according to the environmental and/or circumstantial requirements of an organism, which means that the information is context dependent (Witzany, 2020a).\n\nGroup identity and membership of single RNA stem-loops in ensembles of formerly integrated groups seem to be useful in (re)building new RNA stem-loop groups. We must not forget the essential roles that RNA minorities such as former degraded RNAs play in small RNA stem-loop groups, or even single stem-loops ready to be reused in (re)building current functional RNA groups.\n\nThe genetic code of DNA may represent the result of the effective escape strategy of RNA-networks: out of a competitive RNA world and into a new sequence space to better conserve successfully selected genetic identities. The reverse transciptases and related RNA networks produce complementary strands of RNA to DNA, which means itâs the crucial escape technique. As RNA polymerases they copy a single stranded RNA sequence into a single stranded DNA sequence. The reverse transcriptase is very old and a key driver of genomic plasticity and seems to stem from a retroviral origin (Hu & Temin, 1990; Brosius & Tiedge, 1995; Aziz, Breitbart & Edwards, 2010). In this perspective the DNA world could have startet with a retroprocessing competence, later on transferred to cellular organisms by retroviruses, retrotransposons and related genetic parasites.\n\nThis means DNA can also be investigated as fixed (memorized) evolutionary results arising from RNA stem-loop group competition and cooperation. In this perspective, DNA-based information represents a natural code, and RNA stem-loop groups represent the code editors. This makes sense because if DNA is really a natural code, we must not forget that no natural code codes itself, just as no natural language speaks itself (Witzany, 2014a; Nelson & Breaker, 2017). In all empirical observations to date, there are competent agents that edit, use, reuse or create natural code sequences or natural language-based sequence constructions (Witzany, 2015) Accordingly, the epigenetic markings of such DNA sequences represent how DNA-based information is programmed, to produce proteins and to produce RNAs that regulate the production of proteins according to the contextual requirements. This means that the epigenetic programming determines the meaning (function) of the sequence content to be realized (Nowacki, Shetty & Landweber, 2011).\n\nIn contrast to mathematical modelling, socially interacting RNA stem-loop groups may help us better understand group membership and interactional motifs to establish relevant genetic identities (Villarreal, 2009a; Villarreal & Witzany, 2021) RNA sociology:\n\n⢠may identify the key players that edit (modify and determine) the genetic codes of host genomes as consortia of RNA agents and virus-like genetic parasites that drive evolutionary novelty;\n\n⢠investigates RNA group behavioral motifs and their role in regulating replication, gene expression, recombination, immune functions and generation of new nucleotide sequences;\n\n⢠explains the emergence of new sequence space not as a result of selection arising from error-replication events, but as a result of competent generation of nucleotide sequences and genome editing;\n\n⢠focuses on social behavior of RNA agents with their emerging properties, such as communicative interactions represented by cooperation and competition (Villarreal & Witzany, 2013a; Witzany, 2014b).\n\nThe natural code and its users and their communicative interactions can be investigated as social phenomena which cannot be fully understood by mathematical modelling (see chapter 3) and physical/chemical investigations alone, as these focus on statistical mechanics which differ from biotic behavior (Witzany, 2020b; Villarreal, 2015).\n\n\n3. The outdated 20th century narrative\n\nIn groundbreaking articles Manfred Eigen and Peter Schuster led foundation to the quasi-species concept (Eigen, 1971). In detail they investigated how biological macromolecules, i.e. RNA stem-loop groups interact and self reproduce. They were convinced that the whole object of investigation principally can be fully described in terms of physics, which means, can be computed and depicted in mathematical equations (Eigen & Schuster, 1978). For the authors there was no doubt that biology remains a subdiscipline of physics, and also darwinian principles can be fully understood by formalizable equations. Therefore it was logical to use the formal analogy of quasispecies dynamics and statistical mechanics (Domingo & Schuster, 2016). Biological selection then can be explained as condensation (localisation) of sequence distribution in a limited area in a formal sequence space. Evolution theory would then be based on biochemical kinetics (Biebricher, Eigen & Gardiner Jr., 1985).\n\nEigen subsumed generation of information under a dynamic theory of matter (Eigen & Winkler, 1983). Because reproduction processes in a self-reproducing matter depend on limited energy resources it would be natural that such self-reproducing processes underly a certain error rate which is the basis for variations of a master copy. The evolution of living systems with quasispecies dynamics and proteins depends on an unequivocal âcode-systemâ and a relational protein system which evolves via âhypercyclesâ. This leads to the Eigen-Schuster equations in which evolutionary results are coherent with natural laws of physics (Eigen, 2013). Errors in this system of reproduction are the result of an instability within the system which can be sufficiently explained by irreversibe thermodynamic processes (Eigen, 1971).\n\nEigen refers the explanatory model of the self-reproducing machine to a reality in which these automatons meet the requirements of Darwinâs theory of biological evolution (Eigen & Winkler,1983). He is convinced that without no doubt John von Neumannâs concept of a self-reproducing machine represents a mathematically exact refinement of Alan Turingâs idea of a self-reproducing machine (Eigen, 2013). Some decades later Sydney Brenner argues again that cells and living organisms are good examples of Turing and von Neumann machines (Brenner, 2012). But living nature cannot be properly accommodated within such a theoretical framework (Witzany & Baluska, 2012a). Although proposed for more than 80 years not even one self-reproducing machine has been constructed or seen until today. Empirical data contradict this model of explanation.\n\nEigenâs adaptation of this machine thinking to living agents was a misconception because the language that codes machine programs is not compatible with that of the genetic code. Languages controlling Turing and von Neumann machines are based on formal algorithms, in which syntax determines meaning independently of context (Witzany, 1995). But as epigenetics demonstrated, gene expression essentially depends on environmental context and cannot be similarly treated as a formal language. A formal language syntax which can be expressed by mathematical equations transports unequivocal meanings. In contrast natural languages and codes represent both, a superficial grammar and a hidden deep grammar with varying context dependent meanings (e.g., âThe Shooting of the Hunters.â). Not the superficial DNA sequence syntax determines the meaning (function) but pragmatic context of real life in which organisms are involved. This is a crucial difference in explaining genetic information. In the explanatory models of the 20th century, the superficial DNA syntax was thought to represent the final information not its pragmatic use with its varying context dependent meanings. In contrast to 20th century narratives which tried to identify meaning out of superficial sign sequence syntax, we now know that it is the context in which sign sequences are used that determines the meaning, not the syntax. This was completely ignored by linguist Chomsky and chemist Eigen (Chomsky, 1965; Witzany, 1995). As a consequence, the mathematical equations of the quasispecies concept favoring a mutant spectra out of a fittest master type must be revised accordingly (Villarreal & Witzany, 2013b). It is correct that RNA group interaction motifs âresemble, in many ways, social behaviorâ as proposed by Eigen. But social behavior of living agents does not represent interactions of self-reproducing machines that are programmed by algorithm-based procedures.\n\n\n4. Cellular organisms communicate in any coordination\n\nAfter this clarification of how RNA-groups interact, we now look at cellular life which represents the planetary ecosphere for RNA mediated interactions and regulations. The explosion of knowledge about the early and the current RNA world has not led to a better model explaining how cellular life started out of this RNA world (Manrubia et al., 2021) What we know now is that the essential processes in cellular organisms are regulated by RNA-mediated processes, whether these be the various steps of transcription and translation, DNA repair, immunity, replication and epigenetic markings. In particular, the epigenetic programming of cellular developmental stages is a crucial step in understanding cellular life and designates the epicenter of genetic information (Mattick & Amaral, 2022).\n\nWith the evolution of cellular organisms, a new stage in the evolution of life was reached. Henceforth, stable storage of DNA genetic information was established not only as a blueprint for cellular reproduction processes, but also as a life habitat for an abundance of invading genetic parasites, whether at a persistent stage or a lytic stage (Brookfield, 2005; Le Rouzic, Dupas & Capy, 2007; Vennera, Feschotte & Biemonta, 2009). With the start of cellular life, a completely new dimension originated, that of cellular populations that occupy ecological niches to metabolize and reproduce on this planet. The highly coordinated stages of all essential processes in the cell are stored in the DNA of each cell and are processed according to the different epigenetic markings. In addition, a DNA proofreading mechanism started, which had been absent in the early quasispecies era of RNA viruses and related genetic parasites.\n\nCellular organisms actively compete for environmental resources. They assess their surroundings, estimate how much energy they need for particular goals and then implement the optimum variant. They take measures to control certain environmental resources. They perceive themselves and can distinguish between âselfâ and ânon-selfâ. Current empirical data on all domains of life indicate that unicellular organisms such as bacteria, archaea, giant viruses and protozoa, as well as multicellular organisms such as animals, fungi and plants, coordinate and organize their essential life functions through signaling processes (Witzany, 2010, 2011a, 2012, 2014c, 2017b, 2020c; Witzany & Baluska, 2012b; Witzany & Nowacki, 2016).\n\nSignaling allows for real-life coordination and organization. It is a communicative interaction in which species-specific behavioral patterns and sign repertoires are used according to three levels of rules (not laws) that govern the following: the combination of signs (syntax), the coherence between sign and content (semantics) and, most importantly, the use of signs according to a specific context (pragmatics). Rule-following in communicative interactions is essentially a social event, because a single agent cannot follow a rule only once, as proved by Ludwig Wittgenstein (Wittgenstein, 1953). The use of signs in communicative interactions occurs by social sharing of a common set of signs and a commonly used set of rules.\n\nCells, tissues, organs and organisms communicate at four key levels that are essential for all cell-based life forms (see Figure 2) (Witzany, 2016a, 2019).\n\nThere is no doubt that all atoms and molecules on earth underlie the second law of thermodynamics. The generation of new sequence structures in natural codes, new behavior of organisms, new organs and tissues also underlie these laws. But this does not explain which communicative interactions between RNA networks, viruses and cells lead to new cells, tissues, organs and organisms, because natural laws do not change and adapt but remain fixed (Salmena et al., 2011; Ariza-Mateos & Gomez, 2017; Villarreal & Witzany, 2019). In contrast, communicative rules in generation and combination of signs may change and adapt, which means that biotic innovations may be exapted and coopted for purposes which are different from those which previously emerged (Frenkel-Pinter et al., 2022; Witzany, 2018) The main characteristic of sign-mediated interactions, in contrast to interactions not mediated by signs, is that the proponents may change the rules of sign use for purposes of adaptation, e.g. dialects in bacteria or even in bee languages, and generate really new sign sequences with really new content (Ben Jacob et al., 2004; Schauder & Bassler, 2001; Witzany, 2014d; Baluska & Witzany, 2014). This represents a key feature in the self-empowerment of life.\n\nSelf-empowerment is the essence of biotic communication because it liberates socially interacting agents from the strictly dominating natural laws on abiotic planets and transforms these agents into a sphere of innovation, creativity and productivity to adapt to changing contexts. It is the inherent result of communicating agents, because they can generate really new results by the social use of natural codes, instead of following mechanistically from former conditions (Witzany, 2019). Living agents use signs in natural communication processes, whether as indices, icons or symbols (Atkin, 2010). They combine signs resulting in sign sequences and therefore increase information content and complexity. The contextual sign use by competent agents in populations constitutes meaning in all known natural languages or codes (Prinz, 2022). And sign use in communication processes as well as the emergence of meaning is basically a social event. As previously mentioned, it is not possible that only a single agent invents signs or follows rules.\n\nSigns in biotic communication processes within and between cells may be chemical molecules, electric and tactile signals, or also, as in higher animals, visual and auditive signals. More recently, it was found that cellâcell communication can also be mediated by small RNAs (Lefebvre & Lécuyer, 2017, Buck, 2022) Additionally, abiotic influences, such as gravity, light, temperature, air movement, etc., also serve as signals to organisms which can be sensed, memorized and evaluated and may modify behavioral patterns.\n\nGenetic engineering in the 20th century was dominated by the conviction that DNA sequences can be deleted, modified and inserted like molecular bricks to change the capabilities of certain plants, animals and other organisms. This conviction was supported by the one-geneâone-protein narrative, the perspective that non-coding RNAs represented âjunkâ and the central dogma of molecular biology, that means genetic information flows from DNA via RNA to protein, never reverse. This changed dramatically with the rise of epigenetics. The regulatory system that functions in the development, morphology, cell fate and identity, aging, physiology, genetic instructions, immunity, memory/learning, and physical and mental disease depends on epigenetic marks, DNA methylation and histone modifications (Jablonka & Lamb, 2002; Cabej, 2018). Genetic sequences of all organisms in all domains of life can be marked according to their environmental and social experiences. This means a hidden layer of gene-regulating non-coding RNAs in organisms, which may also be inherited (Jablonka & Lamb, 1992; Mattick, 2003; Shapiro, 2009).\n\nBiotic memorizing is beneficial in helping organisms find better ways of adaptation and to circumvent mechanistic reproduction of âalways the sameâ (Skinner, 2014; Moore, 2016). This forms the basis for learning processes (Zovkic, Guzman-Karlsson & Sweatt, 2013). Learning processes are a teleonomic key technique because they help organisms to react to experiences better than in previous, similar experiences (Crisp et al., 2016; Landires & Núnez-Samudio, 2019; Jablonka, 2013). In the RNA-determined process on the genetic level, we may assume that the purposes of the assembled RNA group processes are directed not toward a singular goal but toward a competence, i.e., a new capability to generate and test innovative tools of adaptation (Marshall & Bredy, 2016; Mattick, 2010; FrÃas-Lasserre & Villagra, 2017; Stajic & Jansen, 2021). This new capability enables organisms to better adapt to environmental circumstances, with various behavioral patterns such as escape, invent, recombine, exapt, coopt, suppress, displace, compete and cooperate and their transgenerational inheritance (Jablonka, 2013; Broecker, 2021).\n\nLiving organisms can adapt to environmental, ecological and random circumstances. This does not mean reproducing the inherited behavioral realms when reacting, but changing this behavior to better fit into a changing âumweltâ (von Uexkuell, 1987) Organisms not only sense and monitor their experiences but even evaluate and compare present experiences with similar ones in the past on the epigenetic level (Shapiro, 2014; McGowan & Roth, 2015). The result is a kind of evaluating comparison (interpretation) of sensory data according to various parameters (Nowacki, Shetty & Landweber, 2011; Casadesús & DâAri, 2002). If the resulting behavior is beneficial in contrast to that of those population members that do not integrate this interpretation, we may term this a successful learning process (Miller & Sweatt, 2007). Learning means sensation, interpretation and adapted behavior, memory and monitoring against stored background information. Learning is a beneficial behavioral motif and outcompetes behavior without learning. Memory is contextual information storage, and learning is the transfer of comparative memory into everyday life (Miller, Campbell & Sweatt, 2008). Without memory, learning is difficult because the comparative data in the background storage are not available. Memory is a pre-condition for learning. With memory and learning, living organisms have an important tool for survival.\n\nSuch memory/learning skills can be found in organisms ranging from viruses and akaryotes to unicellular eukaryotes, fungi, animals and plants, although it was only noted as a capability of higher animals until recently (Csaba, 2017; Baluska, Gagliano & Witzany, 2018). For example, plants can overwrite the genetic code they inherited from their parents and revert to that of their grandparents or great-grandparents. This contradicts the traditional DNA-textbook conviction that offspring passively receive combinations of the genes carried by their parents. Under certain stress experiences, the plant can bypass unhealthy genetic sequences inherited from its parents and revert to the healthier sequences borne by its grandparents or great-grandparents (Lolle et al., 2005; Pearson, 2005; Weigl & Juergens, 2005).\n\nWith the rise of epigenetics, the context-dependent imprinting of experiences at both the phenotypic and the genotypic levels is an essential perspective to understand memory and learning in all organisms. Furthermore, memory and learning depend on a variety of successful communication processes within the whole organism (Jablonka & Raz, 2009; Abraham et al., 2018; Patten et al., 2016).\n\nLearning through memorized experiences to optimize behavior that can be inherited is well documented in all domains of life and does not fit the narrative of chance mutations (error replication) being key evolutionary drivers of genetic variations (Rassoulzadegan & Cuzin, 2015; Spadafora, 2016). We now know that epigenetic switching outcompetes genetic mutations according to the requirements of the environmental context (Gómez-Schiavon & Buchler, 2019; Stajic, Bank & Gordo, 2021). Transgenerational inheritance of such epigenetic memorized experiences opens the door to understanding the emergence of new capabilities which are not the result of replication errors (mutations) (Liberman, Wang, & Greer, 2019; Braun et al., 2020; Fitz-James & Cavalli, 2022). If we look at the roles of RNA networks in the evolution and development of organisms and epigenetic (re-)programming, a new concept appears more appropriate to integrate recent empirical data than the neo-Darwinist approach of the 20th century (Mattick, 2009, 2012; Shapiro, 2022).\n\nCellular organisms primarily try to survive by producing individual and social pathways embedded in situational contexts. This affects bodily impressions, actions and reactions which are relevant for epigenetic markings. At this stage, we have to realize that the transgenerational inheritance of epigenetically memorized and learned capabilities, in particular, does not fit the mutation/selection narrative of the modern synthesis, which is insufficiently complex to integrate that (Witzany, 2021a).\n\n\n5. Bridging the RNA world and cellular life: the virosphere\n\nLast but not least, we have to identify how ancient RNA-world networks came into the cellular domains. Where do all these RNA regulators in cells come from? How did RNA get that crucial role for cell-based life (Herbert & Rich, 1999, Herbert, 2004)? In this respect, it is important to identify those agents that implant the abundance of RNA stem-loops persistently in host genomes (Villarreal, 2012a; Cech, 2012; Lehman, 2015; Atkins, Gesteland & Cech, 2010; Yarus, 2011).\n\nAfter nearly a hundred years of cell biology, it is usual to think about life on this planet as cellular life forms that colonized nearly all ecological niches. All definitions of life have focused on cellular life. We know three domains of cellular life which assemble all kinds of cell-based life, from simple archaea to the most complex brain organs in humans. What appears to be still unusual is thinking about life as a virosphere dominating the whole planet, in so far as viruses outnumber cellular life forms tenfold (Villarreal, 2005; Wolf & Koonin, 2007; Forterre & Prangishvili, 2009). In 1 milliliter of seawater we find 1 million bacteria but ten times more viruses. We must make it clear to ourselves that this planet is a sea of viruses with rare islands representing cellular life. Viruses are clearly more abundant and diverse than their targets, which means that the host cells relevant to the infection are a rare resource within an extremely competitive virosphere (Koonin & Krupovic, 2018; Krupovic, Dolja & Koonin, 2020).\n\nThe early quasispecies, RNA viruses and related genetic parasites can be considered as key drivers of evolutionary processes in the evolution of all domains of life. Viruses have colonized all cellular organisms since the beginning of life (Suttle, 2013; Rohwer & Barott, 2013). In most cases, this is a persistent lifestyle which does not harm the host. Persistent viruses remain as regulatory tools (exapted or coopted), and most of them remain as defectives, which means even fragmented they are effective agents in cellular DNA habitats such as the whole variety of mobile genetic elements and related genetic parasites (Ryan, 2009; Forterre, 2010; Villarreal, 2009b; Roossinck, 2012). We speak about self-splicing introns and an abundance of non-coding RNAs such as SINEs, LINES, Alus, LTRs non-LTRs, retroposons and many others. They may further fragmentize and reassemble, insert and delete, combine and recombine nucleotide sequences without damaging the protein-coding genes which generate the development and growth of cellular phenotypes. They really are masters of natural genome editing in all its steps and substeps (Lambowitz & Zimmerly, 2011; Villarreal & Witzany, 2018; Vignuzzi & Lopez, 2019; Benler & Koonin, 2022; Stedman, 2015; Witzany, 2009). In addition, the various forms of viruses from single-stranded RNA viruses up to double-stranded DNA viruses may combine and recombine their genomic features. Such skills are completely absent in cellular domains of life. Their rich social lives are also coordinated by communication processes (Witzany, 2011b; Erez et al., 2017; Sanjuan, 2021). Persistent infection-derived non-coding RNAs are the main drivers of all single steps in epigenetic imprinting and execution of all related processes (Conley & Jordan, 2012; Slotkin & Martienssen, 2007).\n\nMeanwhile, we know many examples in which viruses and related genetic parasites are key drivers for the evolution of new features in organisms. Well-known examples are the evolution of the placenta of mammals with its retroviral-derived syncytin genes, or the arc proteins, retroviral-derived proteins essential for synaptic plasticity in animals (Bonnaud et al., 2004; Villarreal, 2016a; Day & Shepherd, 2015; Hantak, Einstein, Kearns & Shepherd, 2021). Even the evolution of innate and adaptive immune systems depends crucially on persistent viral infections (Villarreal, 2009c, 2011; Broecker & Moelling, 2019). Furthermore, the split between great apes and humans in their evolutionary history may have been initiated by waves of viral infections (Villarreal, 2004; Witzany, 2021b). Many other examples of viral genome editing by invention and integration of new genes which are currently known include replicase, polymerase, integrase, DNA repair, restriction/modification, methylation, a bilayer nuclear envelope, division of transcription and translation, nuclear pores, tubulin-based chromosome duplication, chitin, calcification, linear chromosomes, cartilage and bones, skin, dermal glands for poison, mucus and milk, larvae, egg and flowering plants (Villarreal, 2005; Atkins, Gesteland & Cech, 2010).\n\nLynn Margulis showed convincingly that the eukaryotic cell is not the result of a series of selected replication errors but a social compound of former free-living prokaryotes (Margulis, 1992). A further question remains the origin of the eukaryotic nucleus, where several new features are present that are completely absent in prokarytic cells but that are present in a variety of DNA viruses (Colson et al., 2018) The precursor of the eukaryotic nucleus may derive from a large double-stranded DNA virus that persistently colonized a prokaryotic host (Villarreal & DeFilippis, 2000; Chaikeeratisak et al., 2017; Takemura, 2020; Bell, 2020) The hosting cell must have lost its cell wall, with the virus incorporating the prokaryotic genes into its pre-nuclear genome, particularly in cases of encoding for metabolism and translation. This virus coordinated both its own replication and its transcription genes. (Interestingly, the α-proteobacteria-derived mitochondria resisted genetic integration and still replicate by themselves but in concert with the host cell.) Such a large double-stranded DNA virus presented a double-layered membrane and a tubulin system. Additionally, such a large DNA virus could integrate the conserved functions of the other unified prokaryotic partners of the eukaryotic cell into a coherent nucleotide line-up of a genetic identity which ensures well-coordinated interacting parts.\n\nA helpful model to understand the persistent lifestyle of viruses and related genetic parasites in cellular organisms is the âaddiction moduleâ. This presents an empirically based explanatory model which may be intrinsic to all regulations/counter-regulations in cellular organisms. Currently we can identify such addiction modules in toxin/antitoxin, restriction/modification and several insertion/deletion modules. They derived from infectious viral clouds and competing viral clouds that, together with the host immune system, reach an equilibrium status which is missing in the members of the species which did not experience these infection waves and their remnants. These addiction modules change the genetic identity of the host organism. Members of the same species that integrated such addiction modules into their genetic identity are protected against the toxin, whereas members without this integration are exposed to the toxin (Villarreal, 2012b, 2016b; Lehnherr, Maguin, Jafri & Yarmolinsky, 1993; Engelberg-Kulka & Glaser, 1999; Kobayashi, 2001; Mruk & Kobayashi, 2014).\n\n\n6. Self-organization of matter and self-empowerment of life\n\nTogether with the RNA remnants of former genetic parasites (defectives), such as long non-coding RNAs and the abundance of short non-coding RNAs or microRNAs and their dominant roles in gene regulation of all cellular processes such as replication, transcription, translation, repair, immunity, epigenetics and additionally the several substeps, we must acknowledge that, without RNA stem-loop group interactions, life as we know it on our planet would not function in detail and as a whole (Mattick et al., 2023). The complementary multiple functions of RNA networks, viruses and cellular life empower life as a whole to adapt and evolve. This means that the early narrative of âself-organization of matterâ should be adapted to âself-empowerment of lifeâ. The step from abiotic physical reactions (âmatterâ) to biotic behavior (âlifeâ) is not a gradual but a fundamental one, because in life we find basic interactional motifs that are really new and which are completely absent on abiotic planets. The new features do not represent gradual extensions or leaps in the quantity of former abiotic features but represent really new ones.\n\nOn looking at the processes where RNA stem-loop group interactions produce random variations, we now have to decide to further term them as a number of errors in replication processes that lead to selection-relevant results. We may now look at such variations as the results of high productivity of random variants. Such variants then become objects to selection processes out of an abundance of varieties of competing and cooperating RNA stem-loop groups. A prominent example are the ribosomal subunits with their different parts, all of which have different historical roots. The inherent high productivity of RNA group behavior then may change the narrative of errors and error thresholds into high productivity and excess productivity. Excess productivity is defined by a limit on the number of base-pairs which a self-replicating RNA stem-loop group may have, before productivity output will not lead to a beneficial increase of information but will destroy the represented information in the following generations of the RNA population. This means that RNA stem-loop groups are able to perform an endless, sometimes opposing and incomputable, number of random productivity results, which are relevant to selection processes and together build the basics for self-empowering life to adapt, reproduce and survive. But if the productivity is too high, self-empowerment decreases.\n\nAlthough high productivity is not a directed process, via biological selection it empowers living agents to adapt and evolve into biological complexity and an abundance of species throughout all domains of life on the genotypic and phenotypic levels (Briones, Stich & Manrubia, 2009; Mercer & Mattick, 2013).\n\nThe differentiation between the molecular biological entities of the 20th century and the socially interacting agents (communication of RNA networks, cells and viruses) based on the 21st century denotes a paradigmatic change in explaining and understanding life (see Table 1).\n\n\n7. Conclusions\n\nThe mechanistic narrative of error replications (mutations) being key drivers of genetic/genomic novelty is insufficiently complex to integrate RNA productivity. This is shown by our current knowledge about the pre-cellular emergence of the RNA world, the dominant role of persistent viruses, their relatives and defectives, such as mobile genetic elements, and their descendants, such as the abundance of non-coding RNAs and their role in gene regulation, generation of variants and genetic novelty, together with knowledge of the variety of epigenetic programming and the transgenerational inheritance of acquired characteristics.\n\nRNA networks, viruses and their defectives constantly produce new genetic variants and generate new sequences which are exposed to selective forces. This constant production is an innovative process, not an error in pre-existing DNA sequences, and therefore should be denoted more precisely as high productivity being the essential driver of random variations. What was described by Manfred Eigen as self-organization of matter, we can now modify and call the self-empowerment of life, because the competencies and behavioral motifs of RNA stem-loop groups empower all organisms which host such groups to evolve, develop, adapt and perform all the regulatory processes necessary for all known life processes. As this high productivity is not part of the modern synthesis of evolution, it is time to develop an integrative theory of evolution which has better explanatory power to integrate the abundance of empirical data on RNA biology, virology and biocommunication of cells produced in recent decades.",
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}
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[
{
"id": "162513",
"date": "10 Feb 2023",
"name": "František Baluška",
"expertise": [
"Reviewer Expertise Cell Biology",
"Plant Sensory Biology",
"Evolution"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is very important and interesting manuscript illuminating diverse roles of RNA networks in evolution and development of organisms in all domains of life. Cooperative evolution of viruses, cells and organisms is under-appreciated but it allows understanding a the most complex issues related emergence and evolution of life. This RNA sociology places life in a correct context and explains many mysteries left behind from the outdated 29th century concepts based primarily on statistical mathematics. Life is not based on mechanistic machines or automatons but is based on truly living organism which actively experience and interpret their ever-changing and very complex abiotic and biotic environment. All organisms are based on cells which communicate, learn and memorize their experiences. I have only three small comments:\n1) Authors could briefly discuss the still confusing issue if viruses are living or non-living agents. Also, why viruses act as living agents only if they invade living cells and seems to be inert (non-living) outside of cells.\n2) For the alternative endosymbiotic origin of the eukaryotic nucleus see Baluška F, Lyons S (2021)1.\n3) Roles of viral infections in the evolution of cells were discussed earlier. Baluška F (2009)2.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "9354",
"date": "20 Feb 2023",
"name": "Guenther Witzany",
"role": "Author Response",
"response": "ad 1: As viruses seem to predate cellular life and together with RNA networks invented cellular life and additionally outnumber cellular life 10 times and infect cells multiple throughout their lifes it would extend energy costs to still carry on cell-independent replication apparatus, which could have led to an evolutionary arms race between cell-dependent and cell-independent replicating viruses. Viruses invented the cellular life-sphere that guarantees their replication. The cellular biosphere on this planet is the dominant lifesphere of viruses. This is the benefit for current viral life styles. We have outlined this definition problem of viruses being alive or not in chapter 6 of: Villarreal LP, Witzany G. Social Networking of Quasi-Species Consortia drive Virolution via Persistence. AIMS Microbiol. 2021 Apr 30;7(2):138-162. ad 2+3: We will insert suggested references"
}
]
},
{
"id": "162510",
"date": "10 Feb 2023",
"name": "Felix Broecker",
"expertise": [
"Reviewer Expertise Mobile genetic elements",
"(evolutionary) virology",
"vaccines",
"immunology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article introduces the concept of \"self-empowerment\" of life, the key message of which is that life forms and their evolution cannot be explained if viewed as purely syntax (coding DNA/RNA) driven automatons (Turing or von Neumann machines). Instead, the specific context (pragmatics) of the syntax, which is governed primarily through RNA agents that communicate via stem-loop structures, is the more important aspect of life. RNAs regulate various life processes including epigenetics (and inheritance thereof), which represents an essential layer of context for the coding genes, which is subject to evolutionary selection and more important for evolutionary processes when compared to random mutations of coding genes. The article is well-written and conveys the message in a comprehensive yet concise manner. Overall the article is already in a very good shape. However, the authors may consider the following suggestions and comments:\nPage 3, around \"An unexpected finding is that cooperative RNA stem-loops outcompete selfish ones\". Looking at experiments and computational data published, for example, by Ichihashi1,2 there is evidence that selfish or parasitic replicators (RNAs) do take over a population, leading to the collapse of the population, unless there is compartmentalization. Once compartmentalization is introduced, not only are the parasites prevented from eradicating the population, they are also actively contributing to the evolution of complexity. I suggest including this information and some relevant references by answering the question whether the models in which \"cooperative RNA stem-loops outcompete selfish ones\" also require compartmentalization.\nPage 4, bottom: Assuming that viruses and cells have common ancestors, that cells evolved from viruses or vice versa, or at least there is constant lateral gene exchange between virosphere and cellular life, do the authors have any explanation as to why there is not a single virus expressing a ribosome? Has the ribosome become too complex (optimized) in present-day cells, requiring dozens of accessory proteins? I am asking also in light of the finding that some of the NCLDVs were found to have genetic information (incomplete) of the translational machinery.\nPage 6, \"The reverse transcriptase (âŠ) seems to stem from a retroviral origin.\" In my understanding, retroviruses as we know them have relatively recently emerged evolutionarily. We know that a ribozyme-reverse transcriptase can evolve3. RTs had existed probably long before the emergence of retroviruses.\nPage 7, \"Empirical data contradict this model of explanation.\" â I suggest softening this to \"There is no empirical data to explain this model.\" (or similar) unless there is actual contradicting data, in which case it should be briefly mentioned/explained.\nPage 7, around the paragraph starting with \"Eigenâs adaptationâŠ\" Very interesting. Just a thought that came to my mind is that, for example, humans and mice share virtually the same set of genes (i.e., the syntax is so similar that you can easily \"knock in\" human genes into mouse cells and they will in most cases perform the exact same functions), yet the phenotype of both species differs substantially. The phenotypic differences are mostly the result of pragmatic context rather than amino acid sequence, i.e., syntax.\nTechnical: There are some full stops missing throughout the manuscript, especially after citations.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "9355",
"date": "20 Feb 2023",
"name": "Guenther Witzany",
"role": "Author Response",
"response": "ad page 3: Yes, we will include these arguments ad page 4: The issue of the ribosome and virus is indeed fundamental and interesting.  Although some viruses do enclose host ribosomes in virions and as noted NCLDV can encode many translational components, to date no virus appears to encode the ribosome itself.  Yet the ribosome itself clearly appears to be a collective of ligated stem-loop RNAs originally derived from various sources (in a virus-like process).  This implies that once the ribosome emerged, it became the fundamental host for all subsequent viruses as well as a fundamental entity for cells themselves.  Thus the clear and lasting virus-host dynamic emerged from the collective and transmissible RNA world and created a host that could acquire meaningful virus-derived code. ad page 6: Yes, important note! As Reverse Transcriptase based early RNA world Replicons predated the DNA world and the cellular world, and retroviruses emerged propably in a later stage of evolution there is no doubt, that retroviruses decended from these early reverse transcriptase sharing RNA replicons. We will note that (see: Koonin et al. Viruses Defined by the Position of the Virosphere within the Replicator Space) ad page 7: Empirical Evidence of the non-existence of von Neumann and Turing machines as well as theoretical considerations contradict this model of explanation. Nature of living organisms cannot be properly accommodated within such a theoretical framework. This is because the language that codes machine programs is not compatible with that of the genetic code. Languages controlling Turing and von Neumann machines are formal algorithms, in which syntax determines meaning independently of context. Gene expression depends on environmental context, however, so cannot be similarly treated as a formal language. ad Page 7, around the paragraph starting with \"Eigenâs adaptationâŠ\": Yes, as most presentations at the meeting in Salzburg 2022 (How Evolution Learnt to Learn) demonstrated the phenotypic differences depend on epigenetic markings, which indeed are the result of pragmatic context of different lifeworlds (of e.g. humans and mice) ad Technical: Will be corrected"
}
]
},
{
"id": "162511",
"date": "21 Feb 2023",
"name": "Ascensión Ariza-Mateos",
"expertise": [
"Reviewer Expertise Structural Biology",
"RNA Structure",
"Virology",
"Hepatitis C Virus",
"RNA-protein Interactions",
"Translation Regulation",
"Fragile X syndrome."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVillarreal and Witzany define a novel and thought-provoking concept in Biocommunications: Self-empowerment as the key feature during evolution and the core of biotic communication. They establish RNA stem-loop groups as essential molecules involved in the initial step between abiotic matter and life. These RNA stem-loops are the main characters of evolution, creating communicative interactions with themselves and other molecules by competition and cooperativity, building molecular consortia, and allowing the emergence of viruses, cells, and organisms. Life complexity is sustained by complex RNA networks, viruses, and molecular editing, constantly changing and adapting to each context, allowing biocommunication at all levels. As experts in Biocommunications, the authors argue for a more complex understanding of the role of RNA productivity, viruses, and epigenetics in shaping genetic variation and driving evolutionary change.\nThe manuscript is thorough, and the authors show a deep and updated knowledge of RNA, Virology, and Evolution, besides providing a compelling and innovative perspective. From my point of view, the article is clear, but the authors assume all readers will have a wide knowledge of Biocommunications, and some ideas are based on concepts not sufficiently described in the text. I understand the difficulties of including all this information, but an introduction to the essential concepts may be helpful for a more fluent reading. Some examples are RNA syntax; âThe Shooting of the Huntersâ; âUmweltâ; Teleonomic key technique; Proponents; Turing, and von Neumann machines ideas; and âhypercyclesâ. Additionally, there is a concept that might be interesting to include in the paper regarding Biocommunication and context: Riboswitches. These are molecules found in all life realms that show conformational changes in response to a specific ligand, like a secondary metabolite, modulating transcription and translation.\nOverall, I consider this study of significant interest to the general readership of F1000Research journal.\nFinally, I would like to give a list of suggestions to improve the clarity and impact of the manuscript:\nFigure 1 provides ample information, but I have found some difficulties trying to understand it. Perhaps more details in the legend will improve its interpretation. Likewise, more details about the flow of arrows should be explained to clarify their meaning. I suggest changing the wording of RNAses to RNases. This is a common misuse, but the proper term is RNase. Section 2.1:\nThe text states that RNase H emerged from the RNA world and is introduced before the proteinsâ emergence. It can be misunderstood as an RNA molecule, but this RNase is formed exclusively by protein. The tri-peptide concept can be ambiguous and unclear for the reader. It would also be helpful to provide some additional explanation. This section is especially engaging and covers a logical evolution in the interconnections between RNA activities and how they became more complex. However, everything is explained as if RNA was the only starter, whereas that occurs in collaboration with other types of molecules which appear during evolution, such as proteins, essential to make more complex relationships with RNA. Maybe the authors mean ribosome and RNase activities in RNA molecules, which evolve later in protein molecules.\n\nSection 2.4:\nThere is a typo, \"startet\" instead of started. Please, consider a period after the reference (Witzany, 2015).\n\nSection 2.5: there is an absence of a period or comma after the references (Villarreal, 2009a; Villarreal & Witzany, 2021). Section 3:\nI have found this section a little hard to understand, and I would appreciate some help from the authors to make it clearer. Especially the sentence:Â The evolution of living systems with quasispecies dynamics and proteins depends on an unequivocal âcode-systemâ and a relational protein system which evolves via âhypercyclesâ. There is a typo, \"irreversibe\" instead of irreversible (include the âlâ).\n\nSection 4: Please, consider a period after (Frenkel-Pinter et al., 2022; Witzany, 2018). Section 5.1: I miss the presence of two periods in this section, after (Colson et al., 2018), and (Villarreal & DeFilippis, 2000; Chaikeeratisak et al., 2017; Takemura, 2020; Bell, 2020).\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "9381",
"date": "27 Feb 2023",
"name": "Guenther Witzany",
"role": "Author Response",
"response": "according the suggestions in the 2nd paragraph of this review: Important issues, but this would need another chapter to integrate all suggestions. further suggestion to Figure 1: will be integrated to RNase H: corrected to section 2.1: looks rather clear to section 2.4: corrected to section 2.5: corrected to section 3, 4 and 5.1: corrected"
}
]
},
{
"id": "162512",
"date": "24 Feb 2023",
"name": "Jordi Gomez Castilla",
"expertise": [
"Reviewer Expertise Biochemistry and Molecular Biology."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis author is in complete agreement and enthusiastic about the change of the narrative of life to a more vital (although not-vitalistic), non-mechanistic and non-only syntactic one. Nietzsche made the first advances in this line for biology taking man as if he were just another biological being where he soon encountered difficulties in thinking in only Darwinian terms.\nâSelf-empowermentâ was exactly the metaphysical alternative set by Nietzsche to confront Spinozaâs âself-preservationâ, the concept that Nietzsche discovered to be the strong metaphysical support to Darwin's natural selection. Malthus' theory was rejected by Nietzsche because its simple to explain the whole life. I agree the âself-empowermentâ concept can be proposed as a hypothesis for scientific research. And it can be formulated in scientific terms, which can be proposed to change M. Eigen's narrative of âself-organization of matterâ, which has self-preservation in its focus. By accepting life as a dynamic of power relations, self-destruction is one of the possibilities. We - men - seem to be moving rapidly in this direction. The hypothesis is that what matters is no longer the individual who enters the struggle of life but winning in the struggle, for which individuals must sacrifice part of their own aspects. This agrees with the authors: from individual replicons, they will become \"quasispeices\" then âconsortia of quasispeices\", covalently-fixed consortia (tRNA), rRNA. Nietzsche, first observed that the struggle of life explained as a competition (a war of all against all) ignores the immense amount of co-operation in the struggle (simultaneous co-operation and competition) because this struggle is not to preserve oneâs life, but rather in the main to achieve power:\n«My idea is that every specific body strives to become master over all space and to extend its force (its will to power) and to thrust back all that resists its extension. But it continually encounters similar efforts on the part of other bodies and ends by coming to an arrangement (âunionâ) with those of them that are sufficiently related to it: thus they conspire together for power. And the process goes on» âDarwin forgot the spirit (that is English!); âŠ.. It will be noted that by \"spirit\" I mean care, patience, cunning, simulation, great self-control, and everything that is mimicry (the latter includes a great deal of so-called virtue).â\nCommunication is at the center for reaching agreements and social relations -âmimicryâ is a communication element- and yes, at the center of self-empowering. Communication rules will change accordingly, the âorganismâ interprets according to its biological interest in surviving and advancing itself. For this reason, Nietzscheâs perspectivism is not entirely relativist, but pragmatic.\nâThere are no facts, only interpretationsâ /// âIn fact, interpretation is itself a means of becoming master of something. (The organic process constantly presupposes interpretations.â\nFoucault and Canetti have also carried out the non-metaphysical formulation of power. In their definitions, Power is the action of a force on another force, and not the action of a force on an object. But, power as such does not exist, it does not accumulate, it is only present as a relation between two or more forces, and it manifests only in action. It normally refers to the government of behaviors.\nMinor points Page 4: âtripetideâ, may be the authors wanted to say dipeptide. Page 7: âsee chapter 3â It is not clear what reference this chapter refers to. Page 9: âSuch memory/learning skills can be found in organisms ranging from virusesâŠâ Add citation of Esteban Domingo. J Virol. 2000 Apr; 74(8): 3543â3547. Memory in Viral Quasispecies. Page 12: Table 1. âdeadâ. Probably better âinertâ?\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": [
{
"c_id": "9389",
"date": "27 Feb 2023",
"name": "Guenther Witzany",
"role": "Author Response",
"response": "Yes, Communication on all levels of sign-mediated interactions within and between RNA networks, viruses and cellular organisms together with epigenetic markings that ensure memory and learning empower biotic agents to survive beyond the outdated concept of mutations (error replications) and selection. Â If better learned skills and competencies are relevant for survival, then it is clear that the old narrative is insufficiently complex to integrate that. An integrative theory of evolution has to outline this in future."
}
]
}
] | 1
|
https://f1000research.com/articles/12-138
|
https://f1000research.com/articles/11-1136/v1
|
04 Oct 22
|
{
"type": "Study Protocol",
"title": "Assessing the impacts of conservation volunteering on participant wellbeing:Â a systematic review protocol",
"authors": [
"Hanna Nuuttila"
],
"abstract": "Background: Volunteers and citizen scientists have become an essential element of most nature conservation and restoration activities due to lack of resources but also due to the wish to engage and interact with local communities where conservation activities take place. Environmental or conservation volunteering is also considered to be a key resource in achieving much needed, ambitious nature restoration programs. Practical conservation work and various forms of environmental enhancement along with recreational and therapeutic use of natural or green and blue spaces have been studied for some time. The value of volunteers and the work is widely acknowledged but few studies have been carried out on the impacts of participating on the volunteers themselves. Using this protocol, a study will be undertaken to assess how impacts of participation have been assessed and reported in the literature; what these reported impacts are; how these are related to reported barriers and motivations for volunteering and whether they are affected by the region or country of study. Methods: This paper will identify studies that have described and assessed impacts of conservation and restoration volunteering on participants at an individual level, with a specific focus on physical, mental or societal wellbeing of individuals. Representative studies were sought from major search engines and relevant stakeholder publications, including both peer-reviewed and âgrey literatureâ in predominantly English language publications, published between 2000 and 2020. A priori inclusion criteria consisted of those publications and reports on studies with volunteer and community participants and which described impacts of, motivations for and barriers to participation. After a critical appraisal, a total of 105 articles were selected for further analysis to provide a narrative and mixed methods synthesis of the evidence base.",
"keywords": [
"conservation",
"volunteering",
"community",
"wellbeing",
"citizen science"
],
"content": "Introduction\n\nWe intuitively know that healthy ecosystems are essential for human wellbeing.1,2 This has become widely accepted and reflected both in international policy initiatives as well as in the vision of major international and national conservation organisations.3 The effect of environmental enhancement along with recreational and therapeutic use of natural or green and blue spaces have been studied for some time.4â6 Nature-based interventions, exposure to natural environments and âgreen exerciseâ have shown to be beneficial to health and wellbeing regardless of age, gender, ethnicity or social status.7 The natural environment is accepted as a vital provider of health and other environmental services whilst life-style related conditions and illnesses influenced by a lack of physical activity, links to natural places, and links to community and people amount to around £180 billion a year in the UK alone.8 The theoretical framework behind this project is based on the Green Mind Theory put forward by Pretty et al., 2017,9 which links the human mind with the brain and body and connects the body with natural and social environments with reciprocal processes: environments shape bodies, brains, and minds; minds change behaviours that shape social interactions and natural capital.\n\nVolunteering and volunteers have become an essential element of most nature conservation and restoration activities due to a lack of resources10â12 but also due to the requirement to engage and interact with local communities where conservation activities take place. Volunteering is acknowledged by UK national governments as a valuable resource to achieve nature conservation and regeneration targets (www.nationalnatureservice.org). The value of volunteers and the work is widely acknowledged but few studies have been carried out on the impacts of participating on the volunteers themselves.13 While many studies report the use of volunteers in conservation projects and describe the value of volunteers to conservation efforts, they rarely describe the impacts of volunteering to volunteers themselves or to the wider community. Qualitative evidence shows that environmental enhancement and conservation activities are valued by participants and contribute to their health and wellbeing, but quantitative evidence is in most part lacking.5,6 As the quality of the evidence is perceived to be low, most reviews as well as individual studies have not been able to draw definite conclusions. Furthermore, few studies have assessed these effects systematically or even described the ways in which these impacts have been assessed.\n\nThis study will review the most recent research on the topic to better understand the impact of conservation activities on participating volunteers, how these impacts of participation have been assessed and reported in up-to-date literature; what these reported impacts are; how these are related to reported barriers and motivations for volunteering and whether they are influenced by the region or country of study. The study will also critically assess the methods used in selected studies to describe and examine the impacts and make recommendations for future studies in the field. As volunteers will continue to be required to support critical nature restoration projects, it is vital to understand how volunteering may impact participants. Furthermore, understanding the positive co-benefits of nature related activities such as conservation volunteering can be used to create pathways for multi-solving interdisciplinary eco-societal issues and fast-tracking these into action.\n\nVarious stakeholders, both academic and non-academic were approached for assistance, including members of the UK health and wellbeing organisation (NHS), volunteer sector, conservation, and community development. Several people were able to assist and contribute to the theoretical framework, question formulation and the literature search strategy, ensuring a thorough understanding of the key concepts relevant to the review.14\n\nOne of the most difficult to define concepts was âwellbeingâ. Here we used a conceptual framework for mental wellbeing, drawing from the literature and practice of the Warwick-Edinburg Mental Wellbeing Scale (WEMWS) in measuring wellbeing.15\n\nThe main objective of this review was to understand whether participation in voluntary conservation and restoration programs has specific impacts on the participants and their communities. The primary research question was:\n\nWhat are the impacts (positive or negative) of voluntary participation in conservation and restoration activities?\n\nIn addition to the primary research question other related questions were identified. These included questions on how the impacts of participation had been defined and assessed; what methods have been used to do so and what variables may affect the extent of these impacts or how they have been evaluated and reported. Conceptual framework for potential impact pathways is depicted in Figure 1.\n\nThe secondary research questions were:\n\n⢠How many studies were identified that specifically describe impacts on participants and/or volunteers?\n\n⢠How many of those impacts specifically describe or refer to the concept of âwellbeingâ?\n\n⢠If âwellbeingâ is discussed, described, or assessed, how has it been defined? What aspects of wellbeing have been included?\n\n⢠How have impacts on participants and/or volunteers been collected, measured, or evaluated?\n\n⢠Has volunteering or participating been shown to have an impact on âwellbeingâ or any of its aspects?\n\n⢠Has participation in conservation/restoration projects been shown to improve any aspect of individual wellbeing?\n\n⢠Has participation in conservation/restoration projects been shown to achieve improved wellbeing for communities?\n\n⢠What motivates people to volunteer?\n\n⢠What barriers have been described which might prevent people from volunteering?\n\n⢠What challenges may arise within volunteering situation that may prevent people from continuing their volunteering commitment?\n\n⢠Where in the world have studies included in this review been conducted? Where are the institutions situated to which the first authors of included studies have been affiliated with? Does this influence types of impacts, barriers or motivations described?\n\nDefinitions of the question components\n\nThe subject/population, or the unit of study was any identified habitat or species conservation or restoration project with a definite aspect of community and/or volunteer participation described in the literature.\n\nThe intervention was the volunteer participation, and the outcome was the effects on participantsâ or perceived mental, physical, social and economic wellbeing, both at individual as well as community level.\n\nThe reviewâs focus was specifically the described (subjective) perceptions of individuals who have participated in volunteering activities. As such, it was not possible to find a suitable comparator, such as review of volunteering impacts from non-conservation projects, that would have provided adequate, reliable data for comparison.\n\n\nProtocol\n\nThe review was conducted following the methodological guidelines set by the Centre for Evidence-based Conservation (CEC) at Bangor, Wales, UK16â18 and the PRISMA-P guidelines.19,20 The study arose from a restricted situation during COVID regulations and is not a standard systematic review in a sense of having resources for a large team of researchers. The study was conducted mostly by one researcher but aiming for the higher standards than that of a typical literature review.\n\nSix bibliographic databases were used for this review: ISI Web of Science, JSTOR, Scobus, ScienceDirect, Google Scholar and Lens.\n\nThe search strings used are listed below (Web of Science format):\n\nâcommunity conservationâ AND âvolunteerâ; âcommunity based conservationâ AND âvolunteerâ; âcommunity-based conservationâ AND âvolunteerâ; âvolunteer conservationâ; âparticipatory conservationâ; âcitizen scienceâ AND âwellbeingâ.\n\nEnglish language was used in bibliographic database searches as well as organizational website searches and web-based search engines. Organisational websites were searched for additional literature, see Table 1. The number of databases used, and the grey literature searched, hopefully ensures the comprehensiveness of the search strategy. Due to lack of resources, there are currently no plans to update the searches during the conduct of the review.\n\nInherent meta-bias is present through the use of mostly English language literature. Although attempts are made in the analysis to identify countries where studies are conducted as well as the nationalities of first authors, it is not possible to disentangle the language bias from the dataset. This will be discussed in the final results.\n\nA total of 13,777 documents were identified from web-based database searches, this figure was reduced to 10,804 after duplicates were removed. An additional 96 articles were sourced from organisational websites, key-literature references and publication depositories and added to the search data (Figure 2).\n\nA priori methodology for screening the articles was conducted in three stages. The initial screening covered only the title of each document, article or a report. Irrelevant titles, as well as all further duplicates were deleted.\n\nA total of 1242 articles and documents remained after the initial title screening (Figure 2). Articles were kept for further reading if deemed otherwise interesting and relevant to the project. The articles and studies identified by their title were downloaded where possible and if not found or the weblinks were unavailable the authors were contacted, and copies requested.\n\nThe checklist against which each article is compared will include the following:\n\n⢠Does the article discuss a conservation/restoration/rewilding project/natural resource management? (YES/NO)\n\n⢠Does it also describe, discuss or assess volunteer participants/community conservation/community participation/citizen science? (YES/NO)\n\n⢠And/or does it also describe, discuss or assess impacts or wellbeing linked to conservation, environment and volunteering in any form? (YES/NO)\n\n⢠Furthermore, does it discuss challenges, barriers, motivations, attitudes, or incentives to such project â anything that would indicate a discussion of the participatory experience, not just merely that volunteers were used to collect data? (YES/NO)\n\nIn addition, included for further reading, were titles that alluded to the above topics which could be discussed in the text, specifically when these were titled as reviews or analyses. Abstracts that related exclusively to ecotourism or tourism were not included.\n\nA total of 272 articles went through an additional abstract screening stage. After which 106 articles remained for more detailed assessment (Figure 2).\n\nA Cohenâs kappa analysis was conducted on a selection of (n=115, 10%) titles between the first reviewer and an additional reviewer to check for consistency of the filtering approach.\n\nFormula used was as follows\n\nK=(Pr(a)-(Pr(e)) / (1-Pr(e)). The resulting Kappa coefficient was 0.7, indicating a moderate to good agreement between the title selection of the reviewer.\n\nThe critical appraisal strategy consisted of assessing the quality or the âinternal validityâ of each study based on the extent, repeatability and clarity of methodology description (âwell describedâ, âlimited descriptionâ, ânot describedâ).\n\nThe generalisability of the âexternal validityâ was ascertained based on the sample size of each study, where studies with less than 20 samples, combined with a limited description of methodology were classified as âlowâ quality (n=2). Studies with 20 or more samples but limited methodological account (n=30) were examined case by case. Initially six of these studies were subjectively given a âhighâ quality classification but this was downgraded to âlowâ after further reflection as no objective, repeatable threshold could be described.\n\nStudies with no description of research methodology were excluded from the outset. Studies were further excluded from the analysis if a low sample size was combined with a methodology that was not adequately described resulting in âlowâ quality data with lack of methodological description. Not all âlowâ quality reports were excluded. Of the final 88 studies, 51 were considered âhighâ quality and 37 âlowâ quality.\n\nThe sample size will be used as a descriptor for the resulting synthesis and processed further when the validity of the evidence base is weighed up and discussed. Reviews were collated for a separate narrative synthesis.\n\nEach article/document was given a meta-data code relating to its database source. All the titles were gathered in Microsoft Excel (Microsoft 365 MSO Version 2207) (RRID:SCR_016137) and assessed according to the critical appraisal strategy. Meta-data extracted included the title, year of publication, country of first author institution, country/countries where study was conducted, and whether these were in âGlobal Southâ or âGlobal Northâ. Data was also gathered to assess whether the article described or assessed: âvolunteer experienceâ, âvolunteer motivationâ, âpositive impacts on or benefits to volunteersâ, âimpacts on the communityâ andâbarriers to or negative impacts on volunteersâ. Not all articles would cover all these topics and in the absence of content, the Excel sheet cell was left blank. Raw data were stored in Figshare.21\n\nThe type of synthesis conducted as part of the systematic review will be a combination of narrative and mixed method syntheses. Descriptive statistics, figures and tables will be used to synthesise the evidence base where appropriate, namely the number, quality, year of publication and geographic spread of articles and other documents collated. This will include assessment of the effect of region and country of the study location as well as the country where the institution or organisation of the first author is based.\n\nFindings will be amalgamated under different themes including:\n\n1) Motivations for volunteering\n\n2) Barriers to participating\n\n3) Perceived impacts from volunteering activities\n\nA narrative, qualitative synthesis form will be used to describe and group different motivations, barriers and impacts identified in the various studies. Not all sources will have covered each of these themes so sample sizes for each will be listed and implications discussed.\n\nThe second part of the study will describe the variety of methods used to gather and assess the above data. Literature reviews, systematic or otherwise, identified in the search phase, will be collated and their findings will be presented using a narrative synthesis. The risk of publication bias will be discussed alongside the strategy to identify potential knowledge gaps and the unrepresented subtopics that may warrant further primary research.18\n\n\nData availability\n\nFigshare: Underlying data for âAssessing the impacts of conservation volunteering on participant well-being: a simplified systematic review protocolâ, https://doi.org/10.6084/m9.figshare.19525615.v1.21\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\nPRISMA-P checklist for âAssessing the impacts of conservation volunteering on participant well-being: a simplified systematic review protocolâ, https://doi.org/10.6084/m9.figshare.21030238.20\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nThe author wishes to acknowledge Suzanne Tarrant, a Consultant Psychologist at Hywel Dda UHB, Dr Liz Morris-Webb at Bangor University and other colleagues at Swansea University researchers in the fields of eco-therapies, blue spaces and well-being, who all provided advice and feedback on conceptual design of the work. The author is also grateful to the team behind the title filtering, Kaisa KÀÀrmemaa and Laura Nuuttila.\n\n\nReferences\n\nGascon M, Zijlema W, Vert C, et al.: Outdoor blue spaces, human health and well-being: A systematic review of quantitative studies. Int. J. Hyg. Environ. Health. 2017; 220(8): 1207â1221. PubMed Abstract | Publisher Full Text\n\nNewton A, et al.: Assessing, quantifying and valuing the ecosystem services of coastal lagoons. J. Nat. Conserv. 2018; 44(February): 50â65. Publisher Full Text\n\nBottrill M, et al.: What are the impacts of nature conservation interventions on human well-being: A systematic map protocol. Environ. Evid. 2014; 3(1): 1â11. Publisher Full Text\n\nBritton E, Kindermann G, Domegan C, et al.: Blue care: A systematic review of blue space interventions for health and wellbeing. Health Promot. Int. 2020; 35(1): 50â69. PubMed Abstract | Publisher Full Text\n\nLovell R, Husk K, Bethel A, et al.: What are the health and well-being impacts of community gardening for adults and children: A mixed method systematic review protocol. Environ. Evid. 2014; 3(1): 1â13. Publisher Full Text\n\nLovell R, Husk K, Cooper C, et al.: Understanding how environmental enhancement and conservation activities may benefit health and wellbeing: A systematic review Environmental health. BMC Public Health. 2015; 15(1): 864. PubMed Abstract | Publisher Full Text\n\nBarton J, Griffin M, Pretty J: Exercise-, nature- and socially interactive-based initiatives improve mood and self-esteem in the clinical population. Perspect. Public Health. 2012; 132(2): 89â96. PubMed Abstract | Publisher Full Text\n\nBerry HL, Bowen K, Kjellstrom T: Climate change and mental health: A causal pathways framework. Int. J. Public Health. 2010; 55(2): 123â132. PubMed Abstract | Publisher Full Text\n\nPretty J, Rogerson M, Barton J: Green mind theory: How brain-body-behaviour links into natural and social environments for healthy habits. Int. J. Environ. Res. Public Health. 2017; 14(7). PubMed Abstract | Publisher Full Text\n\nFoster-Smith J, Evans SM: The value of marine ecological data collected by volunteers. Biol. Conserv. 2003; 113(2): 199â213. Publisher Full Text\n\nCook H, Inman A: The voluntary sector and conservation for England: Achievements, expanding roles and uncertain future. J. Environ. Manag. 2012; 112: 170â177. PubMed Abstract | Publisher Full Text\n\nDickinson JL, Zuckerberg B, Bonter DN: Citizen science as an ecological research tool: Challenges and benefits. Annu. Rev. Ecol. Evol. Syst. 2010; 41(August): 149â172. Publisher Full Text\n\nMcKinley DC, et al.: Citizen science can improve conservation science, natural resource management, and environmental protection. Biol. Conserv. 2017; 208: 15â28. Publisher Full Text\n\nHaddaway NR, Kohl C, da Silva NR , et al.: A framework for stakeholder engagement during systematic reviews and maps in environmental management. Environ. Evid. 2017; 6(1): 11. Publisher Full Text\n\nTennant R, et al.: The Warwick-Edinburgh Mental Well-being Scale (WEMWBS): development and UK validation. Health Qual. Life Outcomes. 2007; vol. 5(no. 64). PubMed Abstract | Publisher Full Text\n\nPullin AS, Stewart GB: Guidelines for systematic review in conservation and environmental management. Conserv. Biol. 2006; 20(6): 1647â1656. PubMed Abstract | Publisher Full Text\n\nHaddaway N, Macura B, Whaley P, et al.: ROSES for Systematic Map Protocols. Version 1.0.2017. Publisher Full Text\n\nJames KL, Randall NP, Haddaway NR: A methodology for systematic mapping in environmental sciences. Environ. Evid. 2016; 5(1): 7. Publisher Full Text\n\nShamseer SL, Moher D, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015; 349(2): 1â25. Publisher Full Text\n\nNuuttila H: PRISMA-P checklist for âAssessing the impacts of conservation volunteering on participant well-being: a simplified systematic review protocolâ. figshare. [Dataset].2022. Publisher Full Text\n\nNuuttila H: ALL original search titles for data depository.csv. figshare. [Dataset].2022. Publisher Full Text"
}
|
[
{
"id": "152400",
"date": "25 Oct 2022",
"name": "Margaret Nohilly",
"expertise": [
"Reviewer Expertise Wellbeing",
"Child Protection"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting article with a focus on volunteers, which as the article outlines is not often the focus of a research project. The article focuses in particular on the impact of conservation activities on participating volunteers. A clear rational is provided for the study and the objectives of the research are clearly outlined.\n\nI recommend that a stronger focus is placed on the concept of wellbeing in this work, particularly in line with secondary research question: If 'Wellbeing' is discussed, described, or assessed, how has it been defined? What aspects of wellbeing have been included? The concept of wellbeing needs to be explored in detail and made accessible to the reader. The conceptual framework outlines a conceptual framework for mental wellbeing. Does this not indicate that mental wellbeing is wellbeing? Mental wellbeing is a component of one of many subsets of wellbeing no more than physical wellbeing, spiritual wellbeing, emotional wellbeing are subsets of wellbeing. I recommend that a more holistic view of wellbeing is explored and presented as a conceptual framework for wellbeing in this article.\nThe protocol for undertaking the work must be commended as it is well detailed and robust.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "9259",
"date": "06 Mar 2023",
"name": "Hanna Nuuttila",
"role": "Author Response",
"response": "I completely agree that I had not defined the concept adequately. I have conducted much more exploration of the concept. The study proposes to understand the ways in which âwellbeingâ has been conceptualised and measured in the field of conservation and following is something I wrote for another paper. I will utilise this to form a more complex, multifaceted definition of the concept for the methods paper. âHuman wellbeingâ can be defined in a variety of ways. As our understanding of wellbeing has evolved in psychology, sociology and economics so has our way of defining and assessing it in conservation practice and policy. Understanding human wellbeing is now a critical component in sustainably managing the environment. Biedenweg et al. identify six human wellbeing indicators (psychological, physical, social, cultural, economic, governance) which are affected by the environment and as such are useful in guiding policy and management (Biedenweg, Stiles, and Wellman 2016). It is generally accepted that human wellbeing is composed of both objective and subjective dimensions (Summers et al. 2012). The objective components are related to material and social attributes and are easily measurable such as our jobs and incomes, homes, places we live in, our environment, available services, infrastructure so on. Equally essential part is the subjective, perceived wellbeing that can only be described by the individual. This relates to job or health satisfaction, our feelings of belonging, empowerment, available choices as well as our subjective measure of mental wellbeing, our relationships and trust in society and civic institutions around us. Understanding of wellbeing has evolved from âbasic human needsâ to encompass ecosystem services, environmental needs and subjective happiness (Summers et al. 2012). We now define âwellbeingâ through how people are feeling and functioning in their everyday lives. Crucially we now acknowledge that wellbeing is a social and cultural construct and aspects of wellbeing can differ drastically geographically, even if some components are universally accepted (Beauchamp et al. 2018; de Lange, Woodhouse, and Milner-Gulland 2016; McKinnon et al. 2016). Â The Office of National Statistics (ONS) in the UK compiles data on ten domains of national wellbeing[1]; personal well-being; our relationships; health; what we do; where we live; personal finance; economy; education and skills; governance and environment. Within these 10 domains there are 44 indicators of national well-being. The indicators include both objective measures (for example, unemployment rate) and subjective measures (such as job satisfaction) to provide a comprehensive picture of the nation's well-being and âsocietal progressâ. In contrast, a wellbeing definition used in a conservation project in a rural Tanzania measures wealth in tropical livestock units, assesses relational and social wellbeing through ability to attend community meetings and places a far more weight and importance in the continuity of traditional ways of life, spiritual practice or female autonomy (Beauchamp et al. 2018). At its most simplistic, human wellbeing in conservation has been defined through three conditions: meeting needs, pursuing goals and experiencing a satisfactory quality of life (Milner-Gulland et al. 2014). Here, we define wellbeing as a multidimensional concept, incorporating all aspects of our lives including social and cultural, environmental and physical, economic and material, and psychological including emotional and spiritual wellbeing. Wellbeing has both objective and subjective aspects. Objective wellbeing is something we theoretically assume can be measured, whereas subjective wellbeing is an externally experienced concept that only the individual can report on. Happiness and wellbeing are sometimes used interchangeably, but constant happiness is not necessarily a prerequisite to oneâs general wellbeing. Here the feeling of happiness is considered as one of the elements of wellbeing â but as a momentary and changeable emotion. It is possible to feel sad and have wellbeing simultaneously, just as it is possible to be of ill health or in inadequate economic situation whilst feel happy or content. This complexity makes measuring wellbeing in its entirety difficult, and is probably the reason why many wellbeing studies aimed at assessing wellbeing focus on limited aspects of wellbeing. [1] Office for National Statistics (ONS), released 11 Nov 2022, ONS website, Statistical bulletin: Quality of life in the UK: November 2022"
}
]
},
{
"id": "157464",
"date": "03 Jan 2023",
"name": "Philip A. Martin",
"expertise": [
"Reviewer Expertise Ecological evidence synthesis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aims to assess the impacts of conservation volunteering on the wellbeing of the participants using systematic review to synthesise the findings of previous studies. This is an interesting question, particularly, as the author states, in light of current attempts to scale up ecosystem restoration.\nI am a specialist in ecological synthesis and have approached my review from this perspective, using the Collaboration for Environmental Evidence checklist for editors and reviewers and a guide (https://environmentalevidence.org/wp-content/uploads/2021/02/CEESAT-Checklist-for-editors.pdf). Based on this, I conclude that this synthesis is likely to be significantly better than the vast majority of synthesis papers published in ecology and conservation. For example it was very good to see (i) a well-defined series of questions; (ii) a flowchart conceptualising the potential impacts of volunteering; (iii) use of a PRISMA diagram; (iv) consistency checks for inclusion criteria; (v) critical appraisal of studies. However, I still think that there are a number of issues that need to be resolved.\nMajor issues\nI am a bit confused about what this protocol is for. Normally, a protocol is written before a study is carried out and pre-registered in order to avoid questionable research practices. However, in the case of this study it seems like much of the study has already been carried out. Is that the case? If so, what is the purpose of this document.\n\nItâs fantastic that a good number of platforms were used to search for relevant literature, however itâs not clear to me what the search strings used for platforms other that Web of Science were. In order to make this work as repeatable as possible it would be helpful if detail on this was provided.\n\nFor the screening, how was it decided that titles were irrelevant? Iâm also unclear as to whether the inclusion criteria were applied at the abstract stage or the full text stage of screening or both. It would be great if you could clarify this.\n\nThe critical appraisal strategy is missing detail. It would be helpful for me if all the of criteria for critical appraisal were presented in a table so it is clearer what the impact of different scores for each category are for the overall scores for internal and external validity.\nMinor issues\nIn the question component section some mention of PICO elements would be helpful.\n\nI canât tell what tool was used for the deduplication of references that were found in searches. Can you please provide some detail on this?\n\nWhy were papers restricted to those published between 2000 and 2020? In addition, although this is mentioned in the abstract, it is not mentioned anywhere in the main text of the article. This information should be included for the sake of clarity.\nTypos\nIn the section on searches âScobusâ should be âScopusâ\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "9258",
"date": "06 Mar 2023",
"name": "Hanna Nuuttila",
"role": "Author Response",
"response": "Major issues I am a bit confused about what this protocol is for. Normally, a protocol is written before a study is carried out and pre-registered in order to avoid questionable research practices. However, in the case of this study it seems like much of the study has already been carried out. Is that the case? If so, what is the purpose of this document.  You are correct. The study protocol was started just after the covid pandemic broke out and left me, the main and only researcher, in a difficult position without much academic support from my institution. The protocol was written but not published for various reasons whilst time and funding to conclude the whole work was running out (this was a short fellowship). I was unable to conclude the intended research as face to face opportunities shrunk and the only thing I could do was to continue with the systematic review â following the guidelines I had written (but not published). As you say â the protocolâs purpose now is just to support work that has already been started â however the findings have not been analysed or written up and all comments and suggestions for the methods will be useful â even if modifications into search methods etc cannot be made. These can, and will be discussed in the research paper â and I must say I have learned a great deal about systematic reviews, protocols and publication.  Itâs fantastic that a good number of platforms were used to search for relevant literature, however itâs not clear to me what the search strings used for platforms other that Web of Science were. In order to make this work as repeatable as possible it would be helpful if detail on this was provided. I have modified and amended this and hopefully this is now clearer: The search strings used are listed below: âconservationâ OR âcommunity conservationâ OR âcommunity based conservationâ OR âcommunity-based conservationâ OR âvolunteer conservationâ OR âparticipatory conservationâ OR âcitizen scienceâ AND âvolunteerâ OR âvolunteeringâ OR âwellbeingâ OR âwell-beingâ In addition, limited searches were conducted for âimpacts of conservationâ, âimpacts of conservation volunteeringâ, âimpacts of community-based conservationâ, âco-benefits of conservationâ, âco-benefits of conservation volunteeringâ, âconservation volunteering AND âhappinessâ, âconservation volunteering AND âhealthâ to check how well the general search string were capturing the intended literature.  For the screening, how was it decided that titles were irrelevant? Iâm also unclear as to whether the inclusion criteria were applied at the abstract stage or the full text stage of screening or both. It would be great if you could clarify this. Title screening consisted of checking the title against the following criteria and whether it alluded that the paper would discuss any of the following: conservation/restoration/rewilding project/natural resource management / volunteer participants/community conservation/ community participation/citizen science / impacts or wellbeing linked to conservation, environment and volunteering in any form / challenges, barriers, motivations, attitudes, or incentives to volunteering or community based conservation or participatory experience. If it wasnât not clear whether or not the article might discuss the above, it was kept for further screening. In addition, I included for further reading, titles that alluded to the above topics which could be discussed in the text, specifically when these were titled as reviews or analyses. I will add the above to the methodology for clarification  The critical appraisal strategy is missing detail. It would be helpful for me if all the of criteria for critical appraisal were presented in a table so it is clearer what the impact of different scores for each category are for the overall scores for internal and external validity. Research quality matrix. Table not visible in this format and will be added to main text. EXTERNAL VALIDITY (sample size) INTERNAL VALIDITY (extent, repeatability and clarity of methodology) < 20 (low) > 20 (high) Not described (no) LOW NO HIGH NO Limited description (lim) LOW LIM HIGH LIM Adequate description (ade) LOW ADE HIGH ADE Matrix results for data used in the analysis. EXCLUDE REVIEW INCLUDE Minor issues In the question component section some mention of PICO elements would be helpful. Below you can see the text from the methods which does identify the PICO elements, I have added codes in brackets for clarity â and repharsed the Main research question with coded elements as follows: What are the impacts (O) (positive or negative) of voluntary participation (I)  in conservation and restoration activities to the participating volunteers (P)? The subject/population (P), or the unit of study was any identified habitat or species conservation or restoration project with a definite aspect of community and/or volunteer participation described in the literature. The intervention (I) was the volunteer participation, and the outcome (O) was the effects on participantsâ or perceived mental, physical, social and economic wellbeing, both at individual as well as community level. The reviewâs focus was specifically the described (subjective) perceptions of individuals who have participated in volunteering activities. As such, it was not possible to find a suitable comparator, such as review of volunteering impacts from non-conservation projects, that would have provided adequate, reliable data for comparison. I canât tell what tool was used for the duplication of references that were found in searches. Can you please provide some detail on this?The âduplicateâ search in Excel was used, as was organising in alphabetical order and visually checking for duplicates. Will add this text to the methods for clarification  Why were papers restricted to those published between 2000 and 2020? In addition, although this is mentioned in the abstract, it is not mentioned anywhere in the main text of the article. This information should be included for the sake of clarity. Thanks, will add this information. 20 yearsâ of datasets was deemed something I could tackle by myself, also In pilot searches the mention of wellbeing was simply not coming up in earlier papers, and knowing Typos In the section on searches âScobusâ should be âScopusâ This will be corrected"
}
]
}
] | 1
|
https://f1000research.com/articles/11-1136
|
https://f1000research.com/articles/12-245/v1
|
06 Mar 23
|
{
"type": "Research Article",
"title": "Effect of problem-based learning on Ugandan secondary school physics classroom practices: an observational study",
"authors": [
"Stella T. Kanyesigye",
"Jean Uwamahoro",
"Imelda Kemeza",
"Jean Uwamahoro",
"Imelda Kemeza"
],
"abstract": "Background: Regular class observations are common ways of monitoring what happens inside classrooms during the teaching and learning processes. From the start of 2020, the Ugandan Education System introduced a new curriculum focusing on active learning methods which foster learner-centered approaches, including Problem-Based Learning (PBL). However, this new curriculum is now only emphasized in the lower classes (grades eight and nine), and teachers of the upper classes are still at liberty to use methods of their choice. This study documented the effect of problem-based learning on Ugandan secondary school physics classroom practices using a Reformed Teaching Observation Protocol (RTOP). Methods: The study followed a quantitative approach with a quasi-experimental design employing cross-sectional survey techniques. The study was carried in 19 secondary schools, both government and private, in Mitooma District, southwestern Uganda. The participants were 419 13th-grade physics students of 2020/2021 school year together with 22 teachers. Professional training in implementing and assessing PBL lessons were offered to some teachers (experimental group), who were compared to those who did not receive the training (control group). The standard and validated international RTOP was used to observe 152 physics lessons. Microsoft Excel 2016 was used to compute descriptive analysis, while IBM SPSS 25 was used to compute inferential statistics. Results: The results indicated that teachers in the experimental group effectively taught their classes using PBL and learners were more active compared to the control group. Female teachers showed a statistically significant difference compared to their male teachers in reformed teaching of physics, while no significance was found between government and private schools. Conclusion: Giving teachers professional training is key to effective classroom practices. We recommend teachers to use PBL in teaching, policymakers train teachers on its implementations, and researchers investigate its effect on other subjects with RTOP or other standard observation protocols.",
"keywords": [
"classroom observation",
"physics class",
"physics teacher",
"problem-based learning",
"Uganda"
],
"content": "Introduction\n\nUntil 2020, the Ugandan education system has basically been using a knowledge-based curriculum. This kind of approach is teacher-centered; usually, teachers tend to focus on getting the syllabus completed, and they are the owners of knowledge, leaving learners to serve as just recipients of Knowledge (Nzeyimana & Ndihokubwayo, 2019). However, students have been found to appreciate more teachers who motivate and involve them during learning (Ukobizaba et al., 2019). Thus, all teachers need to adopt active learning methods such as problem-based learning to enhance problem-solving and critical thinking skills, innovation, and creativity among learners (Ndihokubwayo et al., 2020a).\n\nProblem-based learning is a method comprised of challenging real-life problems with no immediate solution. Learners are first put into groups of five to six members with a chair person and a secretary; they are then presented with a real-life problem by the teacher. They breakdown the problem in order to understand it, devise various ways of solving the problem, choose and implement the best way to solve it, and then they check the appropriateness of the solution (Dorimana et al., 2021). This method engages students actively and equips them with the skills of manipulating hands-on tools, participating in discoveries, and collaborating with their teammates. The teacher, in this regard, acts as an observer, guide, and collaborator, orienting the students to the lessonâs objective (Ndihokubwayo et al., 2020a).\n\nNaturally, people differ in personalities and when participating in the teaching and learning process, they act as individuals. Some of them may even lack up-to-date information concerning appropriate techniques and approaches in dealing with advanced characteristics of teaching and learning (Barrogo, 2020). All these differences in personalities and technicalities in teaching methodologies significantly relate to studentsâ academic achievement as pointed out by Zaare (2013). Additionally, there is a scarcity of documentation of African classroom practices, especially physics teaching in Uganda. Thus, this study employed the Reformed Teaching Observation Protocol (RTOP) to capture and characterize what happens during classroom implementation of problem-based learning.\n\nConventionally, teacher-centered methods have been commonly used during teaching where teachers possess full autonomy over classroom activities (Mpho, 2018) and learners are perceived as mere recipients of knowledge (Karamustafaoglu, 2009). Teacher-centered methods of instruction according to Hill (2002) can be in form of chalk and talk/direct instruction commonly referred to as lecture method. Teachers employing this teaching approach focus on how to organize, structure and present the subject content in a way that makes it easier for the students to understand (Sari et al., 2006); students are required to listen attentively as they assimilate the information (Hill, 2002). The lecture method commonly employed in the traditional teaching over-emphasizes problem-solving instead of conceptual understanding (Richardson, 2004). Students under this method may correctly apply the formula when solving a problem due to memorization but may lack understanding of the basic principles (MiokoviÄ et al., 2012). This approach causes students to lose sight of the educational goals compared to when they are involved in constructing their own knowledge (Mpho, 2018).\n\nWhen researchers want to observe the communicative features of either students or teachers, or both, they can use observation protocols (Vidhiasi et al., 2018). Leary (2013) defines âobservationâ as assessing teaching and learning and developing teachersâ skills and knowledge. They are guides upon which teachers make self-evaluations in order not only to improve classroom practices but also school management as a whole (Zaare, 2013). A classroom observation tool is not meant to threaten teachers but rather to aid them to plan their classroom activities and other professional phases (Barrogo, 2020).\n\nThe RTOP deals with the level to which teachersâ classroom practices match those expected under learner-centred instruction (Sawada et al., 2000). Reformed teaching refers to those classroom practices that lead to high scores on the RTOP (MacIsaac & Falconer, 2002). Formation of the RTOP focused on three major components: learner-centredness, inquiry-oriented, and standards-based (Sawada et al., 2000). This therefore provides the relationship between reformed teaching and the magnitude of learning. Comparing with other observation protocols, RTOP offers the advantage of being inductive and easy to compare with other observation protocols, and different observers can easily confirm it which gives it a greater inter-observer reliability (Sawada et al., 2000).\n\nIn their article, MacIsaac et al. (2001) assessed the practicability of the use of a measurement tool to enhance teachersâ self-reflectiveness. The RTOP tool was employed to understand and define reform teaching. The tool assesses three main pedagogical domains: lesson design and implementation, propositional and procedural knowledge, communicative interactions, and student-teacher relationships. This tool greatly fits the principle followed in this study which focused on science in general and particularly physics, and exclusively on reform rather than general properties including class management, lesson closure, short to administer, high inter-rater reliability, and easily available training and reference manuals (MacIsaac et al., 2001).\n\nAccording to Zaare (2013), handling observation protocols needs one to have a high level of professional ethics and higly objective. During observation, the observer should sit in the back or to the side and should not interrupt the flow of the class. He/she should note down and analyze the beginning activities such as how the room is arranged, and also identify the different areas of the environment which need learners to use specific procedures (Zaare, 2013). They should generally take note of all things seen and heard being aware their prior experiences, personal style, and personal worldview and biases should not be reflected in their perceptions. Observers should try to step back and observe again with an utmost âemptyâ mind, that is, empty of their prejudgments (Zaare, 2013).\n\nBarrogo (2020) analyzed the teachersâ perception of the standardized classroom observation tool, and his results showed that in eight out of the 10 statements, teachers strongly agreed that the classroom observation tool serves as a guide for them to assess their performance and plan for their improvement, thus, enhancing their preparation and competency. In Atkinson and Boltâs (2010) study about using teaching observations to reflect upon and improve teaching practice in higher education, the staff became more interested in undergoing classroom observation as they helped them reflect on their classroom practices. They recommended the process of classroom observation be routine, and that ongoing follow-up should occur (Atkinson & Bolt, 2010).\n\nPiaget (1970) pointed out that children study well by doing as they discover their environment. In this regard, Aderonmu and Obafemi (2015) recommended that in teaching science, learners should be presented with meaningful activities. According to Demirci (2009), and Yilmaz and Ince (2012), learners should be motivated to something in the process of learning rather than only just learning something. The theory of social-constructivism developed by Vygotsky and Cole (1978) thus guided this study. The theory proposes that learners need to create their own knowledge and also be in position to collaboratively use their skills for realization of lesson objectives. Believers of this theory content that since learners have the ability to develop their own understanding via negotiation inside their social setting, teachers thus do not need to transfer knowledge from themselves to the learners. The teacher in this theory is considered to be a facilitator rather than an instructor during teaching and learning process.\n\nThis study was guided by two research questions:\n\n1. Is there a statistically significant difference in physics classroom practices among teachers who use Problem-Based Learning instruction and those employing Traditional methods?\n\n2. Do factors such as teachersâ gender and school type statistically influence physics classroom practices?\n\n\nMethods\n\nA proposal was first developed and approved by the academic committee of the University of Rwanda, College of Education, and was thereafter ethically cleared (Ref: 03/DRI-CE/067/EN/gi/2020) by the universityâs research and innovation office. Permission to conduct research in Uganda was given by the permanent secretary Ministry of Education and Sports â Uganda (Ref: C307/2021). The study did not isolate particular groups, such as those living with HIV or impaired persons, but all grade 13 physics students that belonged to the participating schools were part of the study without any discrimination. A week before the start of data collection, the authors briefed the participants about the aim of the study, and those willing to participate signed written informed consent letters. They were also informed that although publications may arise from the data to be collected, their identities were to be kept anonymous at all times. Thus, no additional consent was obtained for publication purpose and this does not distort any scientific meaning. A few of the participants who were still below 18 years at that moment were asked to first consult their parents, who in turn consented on their behalf by signing the same forms. Each participant was given a code and was referred to only by that code. No monetary compensation was given to participants. Participants were free to withdraw from the study at any time without penalty and were also free not to answer any questions or respond to any research situations if they chose to.\n\nA quasi-experiment research design (Fraenkel et al., 2012) was used in which the participants being observed were either in the experimental group (those trained in the problem-based learning process) or in the control group (those not trained in the problem-based learning process), and the observations took place naturally in the classrooms.\n\nDuring proposal development, the first author approached the District Education Officer (DEO) Mitooma District located in southwestern Uganda and requested for a list of all secondary schools in the district. We have randomly selected 19 secondary schools (Eight government owned and 11 privately owned) at a 95% confidence interval with reference to Krejcie and Morgan (1970). A lotto was then made from which 10 schools were randomly picked and put under the experimental group while those which were not picked (11 schools) formed the control group. All grade 13 physics students in the academic year 2021 participated as cohorts. Physics teachers that happened to be teaching in the schools under the experimental group were then trained in how to implement a Problem-Based Learning (PBL) lesson after which they proceeded with implementing PBL in the physics classrooms. The authors then proceeded to observe the teachers both in the experimental and control groups as they taught. In total, 419 grade 13 physics students together with their teachers were observed. Specifically, 72 observations were made in classrooms instructed by teachers who had been trained in PBL process, while 80 observations were made on classrooms instructed by teachers who did not attend the training in PBL process. The whole class of grade 13 physics students in each of the selected schools participated as cohorts.\n\nData was collected using a Reformed Teaching Observation Protocol (RTOP) as a standardized tool developed by Sawada et al. (2000) and accessible on PhysPort.org. The tool assesses interactive teaching, and it can be used for different levels of education, including graduate, upper and lower secondary, college, among others. At the start of data collection, the interobserver reliability was checked. One lesson was observed by two observers and their ratings were then compared. Across all the 25 RTOP items, an agreement of 96% and 0.82 Kappa statistics was obtained between the two observers. There was no measure of observer error within the RTOP data since no interrater reliability was obtained during the course of the study.\n\nAll the teachers in schools under the experimental group were first trained in how to effectively implement PBL in the classroom, unlike those in the control group. This selective training was to enable authors to assess and document if professional training in PBL had an effect on teachersâ classroom practices. The training given to the teachers in schools under the experimental group was entitled âLearning actively in the classroom: How to use Problem-Based Learning (PBL)â (Kanyesigye et al., 2022c; 2022g). It was aimed at equipping teachers with knowledge of what PBL is, guiding them to generate good PBL questions and be able to conduct a real classroom PBL lesson, and also skill them in assessing PBL lessons.\n\nWhen the authors reported to schools for data collection, the teachers were informed that a scientific study was taking place and that the authors would be attending the lessons with them to observe how teaching was taking place and they were also made aware that the observations would be recorded for purely academic and possibly publication purposes. In total, 152 lessons were observed (72 observations on classes taught by teachers who were trained in PBL and 80 observations on classes taught by teachers who were not trained in PBL).\n\nIn one of the PBL classes, teachers in the previous lesson had presented a problem to the groups: Discuss the fact that knowledge of waves can be used to mitigate energy crisis in Uganda. In one the groups, they formed other sub-questions in their attempt to solve the given problem: What are waves? How is the formation/propagation of waves related to energy generation? What technologies exit that can increase amount of energy generated from waves? So, each of the above questions was researched on and presented by the different members of the groups. They concluded that waves if well tapped can be a potential source of energy (wind energy, solar energy, Hydro Electric Power from water waves, etcetera) hence a remedy to the energy crisis not only in Uganda but worldwide. In most of the classes taught by teachers who did not attend the training in PBL, they would immediately after entering the class start dictating notes to the students. At the end of the sub-topic, they would give guiding questions to the students. But the teachers tended not to follow up on whether the students would answer the given questions or not. Again, Students would also be allowed to ask for clarification during the lesson and the teacher would solely answer the questions raised by the students (raw data can be accessed via Kanyesigye et al., 2022b; 2022f).\n\nWe analyzed data with Microsoft Excel 2016 and IBM SPSS Version 25.0 (open-access alternative statistical software to SPSS is PSPP). Descriptive statistics were used to compute mean scores and standard deviations (STD), while inferential statistics were used to measure the significant difference between control and experimental groups and other variables. Since the data were ordinal data, we changed them into continuous data by averaging all rated data for each RTOP item across several teachers. Descriptive tables and figures were drawn, and these mean scores were taken to SPSS spreadsheets to compute the significance of variables. There were 72 lessons observed in the experimental group and 80 lessons observed in the control group. Prior to computing inferential statistics, we checked if we could use parametric tests such as the t-Test. We tested normality of data and equality of variance, but these assumptions were found to violate the use of parametric tests since Kolmogorov-Smirnov of normal distribution, and Leveneâs Test of equality of variances showed a significant difference (p<.05). Thus, the data were not normally distributed, and variances were not equal across various variables.\n\n\nResults\n\nThis sections simultaneously presents teachersâ demographic information and data related to two research questions: is there a statistically significant difference in physics classroom practices among teachers who use Problem-Based Learning instruction and those employing Traditional methods; and do factors such as teachersâ gender and school type statistically influence physics classroom practices?\n\nDuring the study period, only three students missed one or two lessons due to illness, however, they were able to join back after their health improved. Therefore, all the 419 intended grade 13 physics students together with the 22 physics teachers successfully completed the study. The experimental group (10 schools) consisted of 231 students and 11 teachers, while the control group (9 schools) constituted of 188 students and 11 teachers. Table 1 summaries the demographic information of the teachers.\n\nFrom Table 1, it can be seen that majority of the schools were privately owned in both the experimental (54.4%) and control (63.6%) groups. In the experimental and control groups, 63.6% and 54.5% of the schools participated in both pretest and posttest respectively. Most of the participants (72.7%) in both groups were males; had a teaching experience of 5â10 years (63.6% for experimental and 81.8% for the control group); and had a bachelorâs degree (90.9%) as their highest academic qualification.\n\nFigure 1 displays the score distribution of the experimental group. It shows that the scores skewed on the right side.\n\nLikewise, Figure 2 displays the score distribution in the control group. It can be seen that data are scattered, and the kurtosis is low. From both figures, classroom observation practices are better in the experimental than in the control group because the scores tend to be maximum (4/4).\n\nTable 2 was drawn by computing means and STD between experimental and control groups. The 25 RTOP Statements are from three themes (lesson design and implementation, content, and classroom culture) and five sub-themes: (a) lesson design and implementation (1â5), (b) propositional knowledge (6â10), (c) procedural knowledge (11â15), (d) communicative interaction (16â20), and (e) student/teacher relationship (21â25). It can be seen that teachers who got training in PBL reformed their teaching in the 3.78 and 3.97 range, while those that did not get training in PBL (control group) lowly reformed their teaching. Only two RTOP items among the control group got an average score (>2) and the rest of the scores were below 2. These are item-6 (the lesson involved fundamental concepts of the subject), which scored 3.16, and item-8 (the teacher had a solid grasp of the subject matter content inherent in the lesson), which scored 2.43 out of 4.\n\nFigure 3 displays mean scores for each of the three themes. Each group performed similarly in Lesson design and implementation (items 1â5), Content (6â15), and Classroom culture (16â25), where the experimental group outperformed the control group.\n\nFigure 4 displays mean scores for each of the five sub-themes, and each of the experimental and control groups show a similar performance. For instance, Lesson design and implementation (statements 1â5), propositional knowledge (6â10), procedural knowledge (11â15), communicative interaction (6â10), and student/teacher relationship (21â25) scored each between 3.5 and four. Likewise, the control group scored between 1 and 1.5, except for propositional knowledge.\n\nUsing Independent-Samples Mann-Whitney U Test, it was found that the distribution of mean scores was not the same across categories of teaching interventions (Table 3). Thus, a classroom that was taught with PBL showed a good teaching and learning practices than that taught with the traditional method. The same test showed that the group of students who did both pre-and post-test statistically performed significantly (p<0.001) more than those who only performed post-test.\n\nUsing Independent-Samples Mann-Whitney U Test, a statistically significant difference (p<0.05) between men and women teachersâ classroom practices in favor of female teachers was found. However, the same test showed that the distribution of mean scores was the same across categories of school ownership (Government versus Private schools).\n\n\nDiscussion\n\nThe discussion of the results has been presented in relation to the research questions.\n\nIn this study, a problem-based learning lesson was compared to a traditional instruction method (TIM) lesson using observation tools. PBL showed a statistically significant reformed physics teaching compared to TIM. Several studies have used observation tools to document classroom practices. For instance, MacIsaac et al. (2001) used reformed teaching as a catalyst for self-reflective change in secondary science teaching and found that it possesses a degree of credibility to document teaching and learning activities in a reformed form. RTOP requires a radically new type of teaching, in which the teacher has a radically new role. This is a complete transition from the traditional culture of physics lessons (MacIsaac et al., 2001). The renewed teaching appears quite different from traditional physics courses since the class is no longer focused on the teacher. Our study showed that most RTOP items scored more than 2 out of 4 scores in the class taught with PBL. Thus, this class achieved reformed teaching of mechanical waves because practices occurring in the classroom achieved more than 50% scores (MacIsaac et al., 2001; Ndihokubwayo et al., 2020a).\n\nIn this study, themes and subthemes scored similarly. This was different from other studies. For instance, Ndihokubwayo et al. (2020a) observed the implementation of competence-based learning in Rwandan physics classrooms and found varying scores across the themes of RTOP. They got 48% for content (procedural knowledge) and 60% for classroom culture (studentâteacher relationships). Thus, the uniqueness of our findings is that PBL instruction showed the utmost support to improve studentsâ and teachersâ activities toward active and reformed learning. However, items 6 and 8 showed a good performance even for the traditional class. This was because these items are related to propositional knowledge that any teacher possesses despite the methods he may be using. This is content knowledge learned from school. For instance, the âlesson involved fundamental concepts of the subjectâ shows that any teacher in PBL or TIM class would possess such fundamental concepts of the subject. Likewise, the âteacher had a solid grasp of the subject matter content inherent in the lessonâ shows any teacher, despite the teaching intervention, would be able to grasp such subject matter content knowledge.\n\nIn relation to the findings of Amrein-beardsley and Poppâs (2012), analysis of the performance of subthemes in our study indicated that majority of the participants were previously involved in some behaviors as seen in the classroom culture and pedagogy subscales. What was difficult was how to involve learners in more demanding and rigorous activities and methods. Responses from the survey administered by Amrein-beardsley and Poppâs (2012) indicated that many participants found it difficult to incorporate elements thought to be for only science or math classes, and specifically, those found to be difficulty to transfer to content specific to teacher education. Their study findings showed that use of classroom observations maybe a good guide not only for teachers to reflect on their teaching practices but also for observes to learn from others especially from more successful educators (Zaare, 2013). If a class is observed, the teacher may be enabled to clearly describe their instructional practices, assess inequalities that may exist among students, and also enhance classroom instruction. Generally, the teacher whose class in observed gains in-depth understanding of the theoretical knowledge as well as the practical choices of what, how, and what extent to use that knowledge in the classroom (Zaare, 2013).\n\nThe fact that teachers in PBL class showed a good reform in lesson preparation is that they were trained and were given the opportunity to prepare lessons based on PBL instruction. It is important to plan for lessons before teaching. Various authors concur that lesson planning reflects what the classroom would look like (Ndihokubwayo et al., 2022, 2020b; Nyirahagenimana et al., 2022). Teachers mastered the content as those in TIM class; however, they outperformed them in procedural knowledge. This is related to pedagogical knowledge that an effective teacher possesses. Effective teaching approaches and active learning strategies such as PBL took these teachers to a good level of reformed teaching than those using traditional and passive methods. This shows that if Ugandaâs educational policymakers would let teachers in high school implement a competency-based curriculum (CBC) with an effective pedagogical approach, a good atmosphere would be created in those classrooms.\n\nIn the PBL class, the classroom culture that was both communicative interaction and student or teacher relationships was more observed than in the TIM class. High scores on the RTOP call for student cooperative learning via extended dialogue where by it is the teachersâ role to choose those activities which can facilitate dialogue, and be able to support, guide, and reward studentsâ talk (MacIsaac & Falconer, 2002). MacIsaac and Falconer (2002) highlight that in teaching physics, there is no âgolden roadâ. This implies that on the side of physics teachers, RTOP emphasizes the importance of proper preparation, knowledge, and professional development.\n\nThe pre-test effect improved the data, and classrooms that performed both pre- and post-test performed better than those that sat for only post-test. In fact, the use of Solomonâs four-group design would show these controversies. Analyzing groups that both performed pre-test before intervention and those that only performed post-test remove the effect of pre-test to visualize the effect of teaching intervention. However, since this study is a follow-up of other studies from the first authorâs large doctoral research project, the observation of classroom practices did not aim to document performance and attitude or conceptual understanding (these concepts are already published: Kanyesigye, et al. (2022a, 2022d). The students in a group that performed a pre-test would perform better because they are alerted that they are equipped with a certain intervention, then follow all provided and scheduled activities carefully. However, students with only post-test know nothing until they realize that they perform post-test on activities already done.\n\nSex had an effect in favor of women teachers. The fact that female teachers performed more than their counterparts was a surprising authentic finding in this study since almost no available studies have investigated performance due to gender among teachersâ classroom practices. In a similar study that investigated the performance among pre-service teachers, females showed a better performance than their male counterparts after having been taught with the lecture and lab methods; however, this was the opposite after having been taught utilizing the animation method (Mukagihana et al., 2021). A similar observation did not affect when secondary physics teachers did not visualize different teaching practices (Ndihokubwayo et al., 2020b). Despite the difference that occurred in teachersâ classroom practices; however, no effect of teaching intervention was found in public or private schools. A similar outcome was found during learning optics with physical educational technology (PhET) simulations and videos (Ndihokubwayo et al., 2020b; Uwamahoro et al., 2021). This is a good practice because it shows a good level of Ugandan schools since there is a claim of inequality of schoolsâ quality in various countries.\n\nThis study was limited to schools within Mitooma district which is a rural district located in southwestern Uganda with all secondary schools being both day and boarding. Further studies could be extended to other areas to include those schools that are purely boarding.\n\nThe study was conducted in such a way that all grade 13 physics students existing in a particular school participated as cohorts. These students originate from different areas within the country. More so, recording of visual observations was not directly announced to participants to allow then operate naturally. The observed teachers were both men and women who had professionally trained from different teacher-education institutions and are mandated to serve all over the country. These identified characteristics are believed to be shared by even other students and teachers that were not part of this study. Thus, to a certain extent, the data collected is generalizable.\n\n\nConclusion\n\nWe measured the teachingâlearning practices due to the use of problem-based learning alongside the traditional instructional method among selected Ugandan physics classrooms. We trained teachers on how to use PBL in teaching and learning practices of mechanical waves and observed 152 classrooms. Teachers trained in PBL implemented the PBL instruction in experimental classrooms while teachers untrained in PBL implemented TIM in control classrooms. It was found that teachers who were trained in the use of PBL taught well compared to their counterparts (teachers untrained in PBL). Women teachers showed a statistically significant difference compared to their male teachers in reformed teaching of physics, while no significance was found between government and private schools. We recommended teachers use PBL in high schools in Uganda, policymakers train teachers in its implementations, and researchers investigate its effect on other topics and subjects with RTOP or other standard observation protocols.\n\n\nStudies related to this current study\n\nThe current study is a portion of the corresponding authorâs doctoral research project. There are other studies by the authors related to this study that may overlap in the methods or data. These are:\n\nPublished:\n\nKanyesigye, S. T., & Kemeza, I. (2021). Effect of Problem-Based Learning Instruction on Secondary School Physics Students in Understanding of Electromagnetic Waves. Voice of Research, 10(1), 1â17. http://www.voiceofresearch.org/Doc/Jun-2021/Jun-2021_1.pdf\n\nKanyesigye, S. T., Uwamahoro, J., & Kemeza, I. (2022a). Difficulties in understanding mechanical waves: Remediated by problem-based instruction. Physical Review Physics Education Research. https://doi.org/10.1103/PhysRevPhysEducRes.18.010140\n\nKanyesigye, T. S., Uwamahoro, J., & Kemeza, I. (2022b). Data collected to measure the impact of problem-based learning and document physics classroom practices among Ugandan secondary schools. Data in Brief, 44(108534 Contents), 1â9. https://doi.org/10.1016/j.dib.2022.108534\n\nKanyesigye, S. T., Uwamahoro, J., & Kemeza, I. (2022c). The effect of Professional Training on In-service Secondary School Physics Teachersâ Motivation to Use Problem-Based Learning. International Journal of Learning, Teaching and Educational Research (IJLTER). https://doi.org/10.26803/ijlter.21.8.16\n\nKanyesigye S, Uwamahoro J and Kemeza I. (2022d). Effect of problem-based learning on studentsâ attitude towards learning physics: a cohort study [version 1; peer review: awaiting peer review]. F1000Research 2022d, 11:1240 https://f1000research.com/articles/11-1240/v1\n\nUnder review (at the time of publication):\n\nKanyesigye, S. T., Uwamahoro, J., & Kemeza, I. (2022e). The Impact of Problem-Based Learning on Studentsâ Achievement in Mechanical Waves in Secondary Schools. Research in Science Education (RISE).",
"appendix": "Data availability\n\nWe have previously published a data article in Data in Brief (Kanyesigye et al., 2022b; 2022f ) and full descriptions of the data can be found in this study.\n\nMendeley Data: Data for measuring impact of problem-based learning during learning mechanical waves: MWCS, VASS, RTOP. https://doi.org/10.17632/rdtcgstmps.3 (Kanyesigye, et al., 2022f).\n\nThis project contains the following underlying data:\n\n- Reformed teaching observation classroom practices in Ugandan Secondary Form 6 [Feb-Apr 2021].xlsx (Raw data for the study)\n\nMendeley Data: Data for measuring impact of problem-based learning during learning mechanical waves: MWCS, VASS, RTOP. https://doi.org/10.17632/rdtcgstmps.3 (Kanyesigye, et al., 2022f).\n\nThis project contains the following extended data:\n\n- Ugandan Secondary Form 6 Students Views About Sciences Survey [Feb-Apr 2021].xlsx (Data set on studentsâ attitude towards waves)\n\n- Ugandan Secondary Form 6 Responses on Mechanical Wave Conceptual Survey [Feb-Apr 2021].xlsx (Data set on studentsâ understanding of waves)\n\nMendeley Data: Teacher Training in Implementing Problem-Based Learning. https://doi.org/10.17632/b28d3p7kf8.1 (Kanyesigye, 2022).\n\nThis project contains the following extended data.\n\n- Training of Teachers in Problem-Based Learning.pptx (Literature on how to implement Problem-Based Learning in a real classroom)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe corresponding author acknowledges the financial support provided by the African Center of Excellence for Innovative Teaching Mathematics and Science (ACEITLMS) hosted at the University of Rwanda College of Education (URCE). All authors appreciate the participation of Schools in Mitooma District, Western Uganda, teachers, and students in this study.\n\n\nReferences\n\nAmrein-beardsley A, Popp SEO: Peer observations among faculty in a college of education: investigating the summative and formative uses of the Reformed Teaching Observation Protocol (RTOP). Educational Assessment, Evaluation and Accountability. 2012; 24: 5â24. Publisher Full Text\n\nAderonmu TS, Obafemi DT: Ordeals of Physics Instruction in Nigerian Secondary Schools: Way Forward for the Attainment of Global Competitiveness. Journal of Education and Practice. 2015; 6(20): 87â96. Reference Source\n\nAtkinson DJ, Bolt S: Using teaching observations to reflect upon and improve teaching practice in higher education. Journal of the Scholarship of Teaching and Learning. 2010; 10(3): 1â19. Reference Source\n\nBarrogo SD: Teachersâ Perception of Standardized Classroom Observation Tool. International Journal of Academic Pedagogical Research. 2020; 4(7): 33â37. Reference Source\n\nDemirci C: Constructivist Learning Approach in Science Teaching. HUJ Education. 2009; 37: 24â35. Reference Source\n\nDorimana A, Uworwabayeho A, Nizeyimana G: Examining Mathematical Problem-Solving Beliefs among Rwandan Secondary School Teachers. International Journal of Learning, Teaching and Educational Research. 2021; 20(7): 227â240. Publisher Full Text\n\nFraenkel JR, Wallen NE, Hyun HH: How to Design and Evaluate Research in Education. 8th ed.McGraw-Hill; 2012. Reference Source\n\nHill L: Teaching Methodology. Professional Development Strand. Australian Agency for International Development (AusAID); 2002. Reference Source\n\nKanyesigye ST, Uwamahoro J, Kemeza I: Difficulties in understanding mechanical waves: Remediated by problem-based instruction. Physical Review Physics Education Research. 2022a; 18. Publisher Full Text\n\nKanyesigye ST, Uwamahoro J, Kemeza I: Data for measuring impact of problem-based learning during learning mechanical waves: MWCS, VASS, RTOP. [Data]. Mendeley Data. 2022f. Publisher Full Text\n\nKanyesigye S: Teacher Training in Implementing Problem-Based Learning. [Data]. Mendeley Data. 2022. Publisher Full Text\n\nKaramustafaoglu O: Active learning strategies in physics teaching. Energy Education Science and Technology Part B: Social and Educational Studies. 2009; 1(1): 27â50. Reference Source\n\nKrejcie RV, Morgan DW: Determining sample size for research activities. Educational and Psychological Measurement. 1970; 30: 607â610. Publisher Full Text\n\nLeary MO: Classroom Observation - A guide to the effective observation of teaching and learning. Taylor & Francis; 2013. Issue September 2013. Publisher Full Text\n\nMacIsaac D, Falconer K: Reforming Physics Instruction Via RTOP. Physics Teacher. 2002; 40(8): 479â485. Publisher Full Text\n\nMacIsaac D, Sawada D, Falconer K: Using the Reformed Teaching Observation Protocol (RTOP) as a Catalyst for Self- Reflective Change in Secondary Science Teaching.2001; 4.\n\nMiokoviÄ Åœ, VarvodiÄ S, RadoliÄ V: Undergraduate Engineering Studentsâ Conceptual and Procedural Knowledge of Wave Phenomena. International Journal of Electrical and Computer Engineering Systems. 2012; 3(1). Reference Source\n\nMpho O: Teacher centered dominated approaches: Their implications for todayâs inclusive classrooms. International Journal of Psychology and Counselling. 2018; 10(2): 11â21. Publisher Full Text\n\nMukagihana J, Nsanganwimana F, Aurah CM: How Pre-service Teachers Learn Microbiology using Lecture, Animations, and Laboratory Activities at one Private University in Rwanda. International Journal of Learning, Teaching and Educational Research. 2021; 20(7): 328â345. Publisher Full Text\n\nNdihokubwayo K, Byukusenge C, Byusa E, et al.: Lesson plan analysis protocol (LPAP): A useful tool for researchers and educational evaluators. Heliyon. 2022; 8(1): e08730. PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nNdihokubwayo K, Ndayambaje I, Uwamahoro J: Analysis of Lesson Plans from Rwandan Physics Teachers. International Journal of Learning, Teaching and Educational Research. 2020a; 19(12): 1â29. Publisher Full Text\n\nNdihokubwayo K, Uwamahoro J, Ndayambaje I: Implementation of the competence-based learning in Rwandan physics classrooms: First assessment based on the reformed teaching observation protocol. EURASIA Journal of Mathematics, Science and Technology Education. 2020b; 16(9): em1880. Publisher Full Text Reference Source\n\nNyirahagenimana J, Uwamahoro J, Ndihokubwayo K: Assessment of Physics Lesson Planning and Teaching based on the 5Es Instruction Model in Rwanda Secondary Schools. Contemporary Mathematics and Science Education. 2022; 3(1): 1â10. Publisher Full Text\n\nNzeyimana JC, Ndihokubwayo K: Teachersâ Role and Learnersâ Responsibility in Teaching and Learning Science and Elementary Technology in Rwanda. African Journal of Educational Studies in Mathematics and Sciences. 2019; 15(2): 1â16. Publisher Full Text Reference Source\n\nPiaget J: Science of education and the psychology of the child. Trans. D. Coltman; 1970. Publisher Full Text\n\nRichardson J: Concept Inventories: Tools For Uncovering STEM Studentsâ Misconceptions. Assessment and Education Research. 2004;19â26. Reference Source\n\nSari L, Keith T, Anne N, et al.: How approaches to teaching are affected by discipline and teaching context. Studies in Higher Education. 2006; 31(3): 285â298. Publisher Full Text\n\nSawada D, Piburn MD, Falconer K, et al.: Reformed Teaching Observation Protocal (RTOP). PhysPort.org; 2000.\n\nUkobizaba F, Ndihokubwayo K, Mukuka A, et al.: Insights of teachers and students on mathematics teaching and learning in selected Rwandan secondary schools. African Journal of Educational Studies in Mathematics and Sciences. 2019; 15(2): 93â106. Publisher Full Text\n\nUwamahoro J, Ndihokubwayo K, Ralph M, et al.: Physics Studentsâ Conceptual Understanding of Geometric Optics: Revisited Analysis. Journal of Science Education and Technology. 2021; 30: 706â718. Publisher Full Text\n\nVidhiasi DM, Maritim A, Cilacap N: Classroom observation and research. Jurnal Saintara. 2018; 3(1). Publisher Full Text\n\nVygotsky LS, Cole M: Mind in Society: Development of Higher Psychological Processes. Havard University Press; 1978. Reference Source\n\nYilmaz O, Ince E: The Usage of Alternative Assessment Techniques in Determination of Misconceptions about Electromagnetic Field-Magnetism Contents and Effects of Video-Based Experiments on Stu dentsâ Achievement at Distance Learning Course.2012; 55: 155â160. Publisher Full Text\n\nZaare M: An investigation into the effect of classroom observation on teaching methodology. Procedia - Social and Behavioral Sciences. 2013; 70: 605â614. Publisher Full Text"
}
|
[
{
"id": "304158",
"date": "12 Aug 2024",
"name": "M. Rahmad",
"expertise": [
"Reviewer Expertise sources and technology information"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEffect of problem-based learning on Ugandan secondary school physics classroom practices: an observational study\nsuggestions for improvement\nAbstract The background section should clarify the purpose of the research\nIntroduction Most of the references are out of date and need to be supplemented with adequate, up-to-date references\nMethods In the method of only writing 25 RTOP items, it is better to include the number of items for each main pedagogical domain in the study instrument section.\nThe data collection procedure and explanation of teaching intervention sections should adjust the sentence structure again by writing only the necessary parts.\nResults The results of the study contain sentences that are repetitive from the previous section, such as the number of students and classes. It is better to avoid writing sentences that are repetitive.\nDiscussion Strengthen the analysis of how PBL can show better results in experimental classes than control classes.\nThe discussion needs to be strengthened and sharpened with updated relevant references, so as to clarify the findings of the research\nConclusion conclusion is more dominant in recommendations, It should be adjusted to the objectives or research questions.\n\nRecommendation: The manuscript may need improvement with minor revisions\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "351280",
"date": "28 Dec 2024",
"name": "Ozden Sengul",
"expertise": [
"Reviewer Expertise Physics education",
"scientific practices",
"nature of science"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1- A literature review related to classroom observations and constructivist science teaching and learning should be addressed in the paper. You should define the gap in the literature. 2- Social constructivism requires a definition referring to the scaffolding and internationalization of Vygotsky. 3âThe quasi-experimental design had non-random selection. You said you picked randomly, but there is inconsistency. This should be clarified. 4- How many teachers did you observe? Does sampling include teachers? 5-Did you collect quantitative and qualitative data? RTOP can be used to collect both quantitative and qualitative data. 6- Demographic information can be given in the \"Research Design and Methods\" section. 7- What do descriptive and inferential statistics from the Mann-Whitney U Test explain? Could you please interpret the results? 8- Do you answer the research questions? Do you need to conduct a t-test analysis? 9- Your discussion should explain the strengths and weaknesses of your findings. Do your results address the gap in the literature?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-245
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https://f1000research.com/articles/12-242/v1
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06 Mar 23
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{
"type": "Research Article",
"title": "Impact of economic growth on scientific production in Latin America and the Caribbean based on panel data analysis",
"authors": [
"Oriana Rivera-Lozada",
"Judith Soledad Yangali-Vicente",
"Pablo Alejandro Millones-Gòmez",
"Carlos Alberto Minchón -Medina",
"Tania Valentina Rosales-Cifuentes",
"Judith Soledad Yangali-Vicente",
"Pablo Alejandro Millones-Gòmez",
"Carlos Alberto Minchón -Medina",
"Tania Valentina Rosales-Cifuentes"
],
"abstract": "Background: The great difference in scientific production among countries, especially in Latin America and the Caribbean, may be related to the economic growth of each nation, but countries with larger economies do not necessarily have higher scientific production. Political changes and unstable economies result in little sustainability of scientific production in the countries in these regions. The purpose of this study was to determine the impact of economic growth on scientific production, measured as the variation in the gross domestic product and the number of scientific publications, in Latin American and Caribbean countries. Methods: The analyzed information was collected from the open data source of the World Bank for the years from 2000 to 2018. The analysis was performed using unbalanced data panel models that cross-sectionally considered the countries of Latin America and the Caribbean and longitudinally considered the period 2000-2018 using grouped regression models, fixed effects models or random effects models. The Hausman test was used to choose between fixed and random effects models. Results: Â The results of both the random effects models and the fixed effects models demonstrated the negative impact of economic growth on scientific production. This proves that it is necessary to state alternatives to mend and improve the state of scientific production. Conclusion: Â The present study is relevant because it is one of the first to study the impact of gross domestic product on scientific production in Latin American and Caribbean countries from a longitudinal perspective that also allows evaluating the dynamics of both variables.",
"keywords": [
"growth",
"Scientific",
"production",
"data",
"analys"
],
"content": "Introduction\n\nThe Organization for Economic Cooperation and Development (OECD) reports the entry into the era of knowledge-based economies, which are strictly based on technological innovation, the use of knowledge and information and communication technologies.1 As a result, science and technology have gained great relevance to the current economy. Throughout the years, progress in innovation and research has reached its highest peak, both in scientific productivity and in its complexity2,3 and the idea that the scientific and technological progress of a country is the basis of economic dynamics, production growth and its expansion, is becoming more and more common and more accepted and promoted by international organizations oriented to economic development.4 However, with the presence of COVID-19, this progress and funding have collapsed.5 There were sharp declines in economic growth throughout the world during the first half of 2020. Several major investment banks projected negative global economic growth for 2020 of between -1% and -3% of gross domestic product (GDP), but there was a high degree of uncertainty in these figures.6\n\nBetween January 2019 and January 2020, growth of 2% was expected, but the latest estimates indicate that growth was below 1%. These estimates have changed due to the COVID-19 pandemic. Global stock markets declined as investors began to worry about the economic repercussions of the COVID-19 pandemic. Gross Domestic Product declined, and this pandemic cost the world more than $2 trillion by the end of 2020.7 Although many Latin American countries have seen their economies grow over the last five years, the pandemic has been the catalyst for many sectors to explore ways to overcome it through innovation. As a result, a number of sectors have made significant progress, achieving, among other things, an important economic revival.8\n\nGovernments establish policies that seek to guide the development of their nations, face the structural difficulties that hinder growth options, generate opportunities in the different sectors of society, distribute the wealth generated and thus seek to achieve the highest goals that society has set for itself.9 However, in Latin America, government financial support for research is insufficient and has generated controversy by requiring researchers to publish in high impact journals.10\n\nThe allocation of resources for research in Latin America is greatly disproportionate to that of developed countries, and it is evident that in Bolivia, El Salvador, Guatemala, Paraguay and Peru, the allocation of resources for research and development through public funding and private companies is not a priority for governments.10 Peru is the Latin American country with the fewest resources for research development, specifically compared with the other members of the Pacific Alliance, which also includes Chile, Colombia and Mexico. This is reflected in the low Peruvian representation in the scientific production of the region, where only 1.04% of the scientific publications are by Peruvian authors.11\n\nRegarding the number of publications, Peru has had a slight but continuous increase in spending as a percentage of GDP since 2011 and thus achieved its best result in the last years of the examined period, with 0.12% per year. Consequently, in 2017, Peru ranked seventh in scientific production in Latin America with more than 2700 studies. Mexico published more than 23,000 documents that year, and Argentina published more than 13,000 studies with considerably higher number of researchers.12\n\nLike the rest of the world, in Latin America we must face the COVID-19 pandemic, which highlights the need to invest more in research. It is necessary to have a measurement system that allows us to evaluate the number of scientific publications and the impact of economic growth. These key factors involve the adoption of a solid long-term research policy focused on the allocation of economic resources for research.\n\nResearch and development expenditures include current and capital expenditures (public and private) on creative work undertaken to improve knowledge, including knowledge about humanity, culture and society and the use of knowledge for new applications. Research and development encompass basic research, applied research and experimental development. This seeks to model a scientific and technological culture that involves the collective production of scientific knowledge, interdisciplinary work and the participation of all actors in the field of scientific-technological development of the country.13\n\nHowever, little is known about the impact of economic growth on the number of scientific publications in Latin American and Caribbean countries. Our study identifies the research problem by asking the following question: what is the impact of the GDP on the number of scientific publications in Latin American and Caribbean countries?\n\nThe result of low GDP investment in research in Latin America and the Caribbean is detrimental to innovation, compromising the development of different sectors of the economy, one of the greatest competitive advantages that one country can have over another.14 If a country is interested in improving its competitiveness, it is essential to greatly increase investment in research and development.15 Research aims to create knowledge, strengthen economic growth and implement public policies and social development in countries as a result of their economic development.16\n\nThe policies promoted by governments around the world for the financial support of research have not only boosted the resources available to universities, but have also boosted their academic quality. Evidence of this is the number of researchers working in Science, Technology and Innovation (STI) areas, as there is a record of 7.8 million scientists and engineers working in STI production. This represents an increase of 21% with respect to the number registered by UNESCO in 2007.17 However, in Latin America, the promotion of research in national strategies to improve education, national development and scientific production led by researchers in the different countries of the region remains below the expected values, showing a high dependence on international collaboration to achieve recognition in the international scientific community.18\n\nThe connection between science and economic factors, such as production factors and the construction of the social fabric, was studied by Quinde-Rosales et al.9 These results allowed us to establish a model that demonstrated the bidirectional nature of the GDP and expenditures in science and technology, enabling us to establish that the differences in the two variables are stable and that the expenditure generated by science and technology depends on the GDP.\n\nThe study by Giraldo-Gutierrez et al.19 analyzed the impact of science, technology and innovation (STI) policies on the production and appropriation of knowledge. The results showed that despite the low investment in STI, scientific production has been increasing due to factors such as the creation of research centers and the development of research. Santina and Caregnato20 showed that in different countries, research behaves unevenly. A few countries focus on financial resources and human resources at the internal level and external levels of natural resources and the wide diversity in the integrations of different countries to strengthen research in the countries.\n\nCepeda Avila et al.21 indicate that scientific production has grown over the years in different Latin American countries despite the limitations; however, compared to developed countries, scientific production remains low. The authorsâ explanation for the increase in scientific production is that an increasing number of researchers carry out projects that culminate in publications, although their financial resources are quite scarce. Pérez and Lutsak-Yaroslava22 indicate that scientific production in Latin America is growing and consolidating, as indicated by the increase in publications in high impact journals and the establishment of networks that stimulate the advancement of knowledge.\n\nVargas-Merino and RodrÃguez23 indicate that it is important to continue to increase production capacity and maintain outstanding performance in terms of impact and excellence. The real challenges are to achieve recognition of production led by researchers in these countries, generate true internal capacities for the development of quality research and decrease dependence on international collaboration.\n\nIn view of the above, this research aimed to evaluate the impact of GDP on the number of scientific publications in Latin American and Caribbean countries under pre-pandemic conditions. In this way, it will be possible to determine in the future the countries in which the pandemic had a greater impact on scientific production. Therefore, the objective of our study was to estimate the impact of economic growth on scientific production, measured as the variation in gross domestic product and the number of scientific publications in Latin American and Caribbean countries.\n\n\nMethods\n\nObservational, explanatory, retrospective, cross-sectional and longitudinal.\n\nAll the countries in Latin America and the Caribbean with complete information according to the variables of economic growth and scientific production, in the period 2000-2018. These countries, in alphabetical order, are the following: Argentina (ARG), Bolivia (BOL), Brazil (BRA), Chile (CHL), Colombia (COL), Costa Rica (CRI), Ecuador (ECU), El Salvador (SLV), Guatemala (GTM), Haiti (HTI), Honduras (HND), Mexico (MEX), Nicaragua (NIC), Panama (PAN), Paraguay (PRY), Peru (PER), Dominican Republic (DOM), and Uruguay (URY).\n\nEconomic growth: gross domestic product per capita (GDP, $ at current international prices).\n\nScientific production: articles in scientific and technical journals.\n\nData were downloaded from the open data source of the World Bank (https://datos.bancomundial.org/indicator), considering Latin American and Caribbean countries which have complete information in the period from 2000 to 2018.\n\nThe analysis was performed using static panel data models, considering the Latin American and Caribbean countries cross-sectionally and the period 2000-2018, longitudinally. The initially proposed models derive from the following:\n\nYit = η1i + βXit + uit\n\ni = 1, 2, âŠ, 18 countries t = 1, 2, âŠ, 19 years (period 2000-2018)\n\nWhere, Yit is the scientific output (in natural logarithms, lnarticles); Xit is the GDP per capita (in natural logarithms, lnGDP); uit, the error; ηi and β, the model parameters, with:\n\n(1) Pooled regression model: unobserved heterogeneity\n\nηi = η, constant for all countries.\n\n(2) Fixed-effects model for panel: includes heterogeneity for each country\n\nηi, i = 1, 2, âŠ, 17, different for each country.\n\n(3) Random effects panel data model: unobserved heterogeneity within the error component, as:\n\nÎœit = ηi + uit\n\nModels (1)-(3) are the so-called one-way models, and the conventional assumptions.24,25\n\nOnce heterogeneity among countries and among the years 2000-2018 were verified, we resorted to two-way static panel data models:\n\nYit = ηi + ÎŽt + βXit + uit\n\nWhere ÎŽt captures the heterogeneity associated with time (years).\n\nThe models were compared, and assumptions evaluated with appropriate tests,24,25 using STATA v.16.26 The Hausman test was used to decide between a fixed-effects model and a random-effects model. The estimated two-way fixed-effects model was corrected using cluster-robust estimators by country, in accordance with Hoecche's assessment of assumptions.24\n\n\nResults\n\nThe scientific production in 18 countries of Latin America and the Caribbean, in the period 2000-2018, is shown in Figure 1. This shows that Brazil, Mexico and Argentina have been the countries with the most production, defined as the number of articles published (in neperian logarithms).27 The X axis corresponds to country identification numbers, ordered alphabetically. The lines correspond to the average scientific production of the countries, showing different levels of production that is not captured by classical regression methods (unobserved heterogeneity). It can also be seen that scientific production has changed over time in certain countries more than in others: Argentina has maintained its production over time, and Ecuador has changed more, showing scientific production with different variance between countries.\n\nKey: Argentina (ARG), Bolivia (BOL), Brazil (BRA), Chile (CHL), Colombia (COL), Costa Rica (CRI), Ecuador (ECU), El Salvador (SLV), Guatemala (GTM), Haiti (HTI), Honduras (HND), Mexico (MEX), Nicaragua (NIC), Panama (PAN), Paraguay (PRY), Peru (PER), Dominican Republic (DOM), and Uruguay (URY).\n\nFigure 2 shows the relationship between the impact of economic growth and scientific production, in which we can see, through the solid line, the general association and by each country of Latin America and the Caribbean. There is evidence of a positive trend. At country level, less impact is observed in comparison with Latin America and the Caribbean.\n\nPooled regression model (OLS). Key: Argentina (ARG), Bolivia (BOL), Brazil (BRA), Chile (CHL), Colombia (COL), Costa Rica (CRI), Ecuador (ECU), El Salvador (SLV), Guatemala (GTM), Haiti (HTI), Honduras (HND), Mexico (MEX), Nicaragua (NIC), Panama (PAN), Paraguay (PRY), Peru (PER), Dominican Republic (DOM), and Uruguay (URY).\n\nThe proposed static panel data models were the following: pooled regression, fixed effects and random effects. The estimated models, shown in Table 1, confirm the expected impact of economic growth on scientific production, which is estimated to be positive, Î²Ì = 2.91256 (p < 0.05) in the pooled regression model (OLS), Î²Ì = 1.77793 (p < 0.05) in the fixed effects model, and Î²Ì = 1.78803 (p < 0.05) in the random effects model; the coefficients of determination were 50.43%, 69.4%, and 69.4%, respectively. The first order correlations (rho) were estimated to be 0.971 and 0.967 in the fixed and random effects models, respectively. Apparently, the pooled regression model indicates a greater impact (see OLS in Figure 2), but, as we know, it does not consider differences between countries (unobserved heterogeneity).\n\nThe models were compared using various tests. In Table 1, heterogeneity between countries is evident (F = 550.05, p = 0.0000 < 0.05), unobserved by the pooled regression model. On the other hand, the Hausman test (Chi-square = 3.29, p = 0.0695 > 0.05) indicates no difference in the impacts estimated by the fixed effects model and the random effects model. Likewise, the Breusch and Pagan Lagrange multiplier test for random effects (Chi-square = 2674.65, p = 0.0000 < 0.05) indicates that the random effects model is better than the pooled regression model.\n\nThe assumptions of the fixed effects model were also evaluated. The Breusch-Pagan LM test for errors independence between countries results in its rejection (Chi-square = 790.614, p = 0.0000 < 0.05), which establish the dependence between them. First order autocorrelation was evaluated with the Wooldridge test (F = 16.683, p = 0.0008 < 0.05) and the Baltagi-Wu LBI test (rho = 0.634, F = 62.44, p = 0.0000 < 0.05), which show the presence of first order autocorrelation in both tests. The heteroscedasticity of the errors, using the modified Wald groupwise heteroscedasticity test for countries (Chi-square = 4798.87, p = 0.0000 < 0.05), indicates different variabilities between countries.\n\nOnce the heterogeneity of scientific production between countries was verified, the possible heterogeneity over time was also evaluated. Figure 3 shows a positive trend in scientific production in each country. Colombia, which in 2000 produced less than Chile and Argentina, in 2018 equaled Chile, and the production in both was close to Argentinaâs production. Ecuador, whose production in 2000 was lower than Costa Ricaâs, Uruguay and Peru, surpassed them as of 2016. In summary, average scientific production is expected to increase over time, showing temporal heterogeneity, giving rise to two-way models. The models estimated in Table 1 correspond to one-way models.\n\nKey: Argentina (ARG), Bolivia (BOL), Brazil (BRA), Chile (CHL), Colombia (COL), Costa Rica (CRI), Ecuador (ECU), El Salvador (SLV), Guatemala (GTM), Haiti (HTI), Honduras (HND), Mexico (MEX), Nicaragua (NIC), Panama (PAN), Paraguay (PRY), Peru (PER), Dominican Republic (DOM), and Uruguay (URY).\n\nThe two-way estimator models are shown in Table 2, including fixed-effects and random-effects panel data models. Compared to 2000, scientific production was differentiable in almost all years, with the exception of 2001 and 2002, when it was similar, both in the fixed effects model (p = 0.318 > 0.05 and p = 0.052 > 0.05, respectively) and in the random effects model (p = 0.349 > 0.05 and p = 0.068 > 0.05, respectively). Heterogeneity between countries is ratified (F = 850.61, p = 0.0000 < 0.05), and temporal heterogeneity is accepted (Chi-square = 169.59, p = 0.0000 < 0.05) through an additional test. The coefficients of determination improved, to 81.52% in the fixed effects model, and 81.45% in the random effects model.\n\nThe observed heterogeneity in the estimated two-way panel data models shows that the estimated impact of economic growth on scientific production, contrary to what was expected, is negative Î²Ì = -0.55548 (p = 0.002 < 0.05) in the fixed effects model and Î²Ì = -0.36630 (p = 0.041 < 0.05) in the random effects model.\n\nThe Hausman test establishes differences in the estimation of the parameters between both two-way models (Chi-square = 20.87, p = 0.0000 < 0.05), including differences in the impact of economic growth, indicating that the fixed effects model is adequate. Likewise, the Breusch-Pagan LM test for errors independence between countries showed their dependence (Chi-square = 643.664, p = 0.0000 < 0.05), and the modified Wald groupwise heteroscedasticity test (Chi-square = 2520.56, p = 0.0000 < 0.05) confirmed their presence. This situation led us, following Hoecche, to resort to the use of robust estimators by clusters to correct these drawbacks.\n\nThe two-way fixed effects panel data model with robust estimators corrects the standard errors of the estimators and modifies their significance level. In general, the model maintains the similarity of scientific production in 2001 and 2002 compared to 2000, and the differences in subsequent years.\n\nAs for the impact of economic growth on scientific production, estimated by the robust method, although it continues to be negative (Î²Ì = -0.55548, sÎ²Ì = 0.33876), it does not reach statistical significance (p = 0.119 > 0.05), which indicates the absence of such impact, and not the expected positive impact in Latin American and Caribbean countries included in the study.\n\n\nDiscussion\n\nThe aim of this study was to evaluate the impact of economic growth on scientific production in Latin American and Caribbean countries in order to collect important basic data to respond to the problems of low scientific production due to economic and structural causes faced by the region. This is important because one of the main objectives of the countriesâ spending on research is to achieve higher-level scientific results that can improve the populationâs standard of living.28\n\nOne of the main findings was the absence of impact of economic growth on scientific production by the robust method, since this result differs from that reported by many studies that found a relationship between both variables.29â33 However, it is likely that this difference is due, in the first place, to the characteristics of the study itself and to the fact that the evidence reported on the relationship between GDP and scientific production are, for the most part, specialized studies that include publications worldwide and do not cover scientific production in general.\n\nFor example, Jaca et al.33 published a global bibliometric analysis of publications on vaccine refusal in the last 45 years, finding a relationship between scientific productivity and GDP. In addition, Latin American and Caribbean countries had the lowest production rates compared to the United States and European countries, which has also been reported by Ibañez-Marti.34 Another example is reported by Senel in 2020,35 who found an association between GDP and scientific productivity in the countries, in addition to a low contribution by Latin American and Caribbean countries in the area of immunology. In contrast, Ronda-Pupo reported a growth in global scientific production in the Latin American and Caribbean region36 although, when productivity is analyzed by specialties or areas of knowledge and compared with the rest of the world, this region is below the average.37\n\nThe impact of economic growth on scientific production in Latin America and the Caribbean for the period 2010-2018 did not differ broadly among countries. Economic growth was maintained in several countries; however, it mainly decreased in Brazil, Chile, Ecuador, Panama and Peru, and it decreased from 2012-2013 in Colombia and Venezuela. Latin America and the Caribbean continue to be a region with average or lower-than-average performance for most knowledge indicators and most growth components.38 This may be due to its primarily product-based economy that is focused on the export of materials and unprocessed goods and has little added value from knowledge and technology, which creates a development gap in advanced technology that affects its economy. No less important is the conduct of research that does not respond to the priorities of policy-makers and that is not aligned with the needs of those responsible in the real world.39\n\nIn terms of scientific production, Brazil and Mexico were the frontrunners, which coincides with what has been reported by other authors, such as Tibaná G.,40 Ronda-Pupo36 and Ibañez-Marti.34 This leadership on the part of Brazil is probably due to the fact that, according to Tibaná, it is the only country in the region with an investment of more than 1% of GDP in scientific research. Moreover, with the exception of Brazil, there are few countries in Latin America that promote research and development activities with an intensity comparable to that recorded in developed countries.41\n\nBesides being the country with the highest growth rate, this investment in research allows Brazil to have more than 400 journals in Scopus and to be ranked 15th in the world. It is worth mentioning that the areas with the highest scientific production correspond to ecology, technology and health.42 However, Ozsoy and Demir43 reported in 2017 that Brazil is the country with the highest scientific production on bariatric surgery worldwide, leaving behind countries such as the United States or China, providing strong evidence of inequality in science in the region.\n\nThis could be indicative enough to conclude that science in the region is marked by a strong inequality, led by few countries, characterized by low regional integration20 and with a high intercontinental participation led by Brazil.44 In the case of Mexico, 68.13% of the articles that received funds from CONACYT were published in restricted-access journals. This means that a large part of the Mexican population does not have access to the results of research financed with state resources. According to economic theory, this restricted circulation of knowledge can undermine the advancement of basic science and innovation.45 On the other hand, scientific production in Brazil remained the same, despite the fact that in recent years, Brazilian scientists have faced a drastic reduction in financial support for research and graduate programs.10 According to JarrÃn-V et al.,28 the efficiency with which a country assimilates investment in research and development depends on (and is limited by) its installed capacities (scientific infrastructure, human resources, programs and laws, etc.). Argentina, Colombia and Chile were the countries with the highest scientific production in the 2010-2018 period. However, Colombia, Costa Rica, Chile, Ecuador, Guatemala, Honduras, Paraguay and Peru also showed consistent growth, though to a lesser degree than the leading countries. Although Latin America has increased its number of scientists and research institutions in recent years, the gap between developed countries and Latin American countries is alarming.37 The primary importance of science and technology for the development of a nation remains unrecognized. The main factors that contribute to low scientific productivity are limited access to grant opportunities, inadequate budgets, deficient levels of infrastructure and laboratory equipment, the high cost and limited supply of reagents and the inadequate salaries and personal insecurity of scientists. Political and economic instability in several Latin American countries has translated into a lack of the types of long-term goals that are essential for the development of science. In Latin America, science is not an engine of the economy.46\n\nThe results in this study of the panel data regression were used to classify countries according to their assimilation of investment in research over time.45 The role of the university in training professionals and generating knowledge through the development of research is key to a countryâs economic growth. Therefore, at the university level, there must be an appropriate economic and institutional approach, adequate information infrastructure, a solid base of human capital and an efficient national information system.47,48\n\nOur study proposes a useful model panel data models that consider the differences in the impact of economic growth on scientific production in countries, fixed or random. This model makes it possible, with large samples and few assumptions, to estimate functional relationships between two variables that provide evidence of the importance of science and technology for academic, economic and social development.49â52\n\nAlthough the purpose of this research was to show a broad panorama of the countries of Latin America and the Caribbean, it is important to note that each nation has intrinsic characteristics that can affect its economic growth and its scientific production. However, all of them require the implementation of national policies that converge into a cooperative process that transforms knowledge into wealth and makes these countries more competitive against developed economies in terms of private sector participation in research and development activities. This requires policies that encourage private investment in an environment that is more favorable to new long-term investments. As most of the investment in research and development at present comes from the public sector, it is strategic to convert private resources into knowledge in the effort to consolidate innovation potential.\n\nThe present study has some limitations: (1) only countries with complete information in the World Bank's open database were included, so many countries were omitted, which can generate important losses of information; (2) measuring the scientific production of a country can be complicated since it can be understood differently according to personal perception. In addition, the impact of such an indicator has not been taken into account, such as the H index or type of journals, which could lead to different conclusions, since although there may be greater scientific production in a given time interval, this does not necessarily mean that there is greater impact of such publications. However, this is one of the first studies that seeks to relate economic growth and scientific production, and therefore provides important information that will serve as a basis for subsequent studies.\n\n\nConclusion\n\nThe present study is relevant because it is one of the first to study the impact of gross domestic product on scientific production in Latin American and Caribbean countries from a longitudinal perspective that also allows evaluating the dynamics of both variables. It was found that Brazil and Mexico lead the scientific productivity indexes in the region, while countries such as Peru, Chile and Uruguay have been increasing their scientific production but remain below the average in comparison with the leaders in the region and the world. However, it is necessary to carry out more studies on the subject since scientific production can be studied from different approaches including indicators such as the impact of scientific publications, the type of journal where the countries of the region publish the most and the level of international and intercontinental contribution in these publications. Therefore, a better understanding of the subject will allow better decisions to be made for a better management of national resources directed to research and development.",
"appendix": "Data availability statement\n\nZenodo: Oriana Rivera-Lozada, Judith Soledad Yangali-Vicente, Pablo Alejandro Millones-Gòmez, Carlos Alberto Minchón Medina, & Tania Valentina Rosales-Cifuentes. (2022). Impact of economic growth on scientific production in Latin America and the Caribbean based on panel data analysis. https://doi.org/10.5281/zenodo.7272492. 27\n\nThis Project contains the following underlying data:\n\n- Growth economic.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0 International).\n\n\nReferences\n\nOECD: New indicators show that the knowledge-intensity of OECD economies is increasing. ParÃs: 2001.\n\nBornmann L, Mutz R: Growth Rates of Modern Science: A Bibliometric Analysis Based on the Number of Publications and Cited References. J. Am. Soc. Inf. Sci. Technol. 2014; 64: 1852â1863. 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Publisher Full Text\n\nStataCorp: Stata Statistical Software: Release 16. College Station; 2019.\n\nRivera-Lozada O, Yangali-Vicente J, Millones-Gòmez P, et al.: Impact of economic growth on scientific production in Latin America and the Caribbean based on panel data analysis.2022. Publisher Full Text\n\nJarrÃn-V P, Falconà F, Cango P, et al.: Knowledge gaps in Latin America and the Caribbean and economic development. World Dev. 2021; 146: 105602. Publisher Full Text\n\nZyoud SH: The Arab regionâs contribution to global COVID-19 research: Bibliometric and visualization analysis. Glob. Health. 2021; 17: 10â31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFan G, Zhou Z, Zhang H, et al.: Global scientific production of robotic surgery in medicine: A 20-year survey of research activities. Int. J. Surg. 2016; 30: 126â131. PubMed Abstract | Publisher Full Text\n\nUgolini D, Neri M, Casilli C, et al.: A bibliometric analysis of scientific production in mesothelioma research. Lung Cancer. 2010; 70: 129â135. PubMed Abstract | Publisher Full Text\n\nMohebi S, Parham M, Sharifirad G, et al.: Social Support and Self - Care Behavior Study.2018; 1â6. Publisher Full Text\n\nJaca A, Iwu-Jaja CJ, Balakrishna Y, et al.: A global bibliometric analysis of research productivity on vaccine hesitancy from 1974 to 2019. Hum. Vaccin. Immunother. 2021; 17: 3016â3022. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIbañez-Marti JJ: La Ciencia en Latinoamérica: Tendencias y patrones. Rev. Fac. Ciencias. 2018; 7: 23â39. Publisher Full Text\n\nÅenel E: The depigmented literature: A holistic analysis of global vitiligo publications between 1975 and 2017. Indian J. Dermatol. 2020; 65: 388â395. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRonda-Pupo GA: Producción cientÃfica e impacto del sistema de ciencia de Latinoamérica y el Caribe en revistas de la región. Investig. Bibl. Arch. Bibl. Inf. 2021; 35: 45. Publisher Full Text\n\nNaciones Unidas: América Latina y el Caribe rezagada en investigación y desarrollo. Comun prensa. Comisión Económica para América Latina y el Caribe.2004.\n\nLanglois EV, Mancuso A, Elias V, et al.: Embedding implementation research to enhance health policy and systems: A multi-country analysis from ten settings in Latin America and the Caribbean. Heal. Res. Policy Syst. 2019; 17: 17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUgarte-Pineda E, Parra-Huerta G: La importancia del financiamiento sobre la producción cientÃfica en México. Investig. Bibl. 2021; 35: 187â202. Publisher Full Text\n\nTibaná-Herrara G: Latinoamérica: producción cientÃfica y tendencias de crecimiento â Scimago Lab Blog. SCImaho Lab; 2021.\n\nOrganización de las Naciones Unidas para la Educación la Ciencia y la Cultura (UNESCO): Panorámica Regional: América Latina y el Caribe. Informe del Seguimiento de la EPT en el mundo 2011. Una crisis encubierta: conflictos armados y educación; 2010; 1â20.\n\nCubana R, Militar DM: Producción cientÃfica en América Latina y el Caribe en el perÃodo 1996-2019 Caribbean in the period 1996-2019.2020; 49: 1â9.\n\nOzsoy Z, Demir E: The Evolution of Bariatric Surgery Publications and Global Productivity: A Bibliometric Analysis. Obes. Surg. 2018; 28: 1117â1129. PubMed Abstract | Publisher Full Text\n\nVélez-Cuartas G, Lucio-Arias D, Leydesdorff L: Regional and global science: publications from Latin America and the Caribbean in the SciELO Citation Index and the Web of Science/Ciencia regional y global: publicaciones de América Latina y el Caribe en el SciELO Citation Index y la Web of Science. El. Prof. Inf. 2016; 25: 35â46. Publisher Full Text\n\nOliveira EA, Martelli-Júnior H, Silva ACSE, et al.: Science funding crisis in Brazil and COVID-19: deleterious impact on scientific output. An. Acad. Bras. Cienc. 2020; 92: e20200700âe20200703. PubMed Abstract | Publisher Full Text\n\nHayne LA, Wyse ATS: Econometric Analysis of Brazilian Scientific Production and Comparison with BRICS. Sci. Technol. Soc. 2018; 23: 25â46. Publisher Full Text\n\nRoldan-Valadez E, Salazar-Ruiz SY, Ibarra-Contreras R, et al.: Current concepts on bibliometrics: a brief review about impact factor, Eigenfactor score, CiteScore, SCImago Journal Rank, Source-Normalised Impact per Paper, H-index, and alternative metrics. Ir. J. Med. Sci. 2019; 188: 939â951. PubMed Abstract | Publisher Full Text\n\nKlepa TC, Pedroso B: Analysis of the technical-scientific production of the National Council for Scientific and Technological Development (CNPq) productivity fellows in Pediatrics. Einstein (São Paulo). 2019; 18: eAO5043. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlzate-Pamplona F-A, Cuartas-Arias M: A contribution to the Latin Americaâs Scientific Production and Edition. Int. J. Psychol. Res. 2020; 13: 9â13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVera-Monge V, GarcÃa-Yu I, de-la-Cruz-Oré J, et al.: Producción cientÃfica latinoamericana en economÃa de la salud, 2005-2014. Salud Publica Mex. 2017; 59: 505â506. Publisher Full Text\n\nMillones-Gómez PA, Yangali-Vicente JS, Arispe-Alburqueque CM, et al.: Research policies and scientific production: A study of 94 Peruvian universities. PLoS One. 2021; 16: e0252410. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLira LAN, Vicente JSY, Huaita DMA, et al.: Factors associated with the development of research skills in graduate students. Universidad Cesar Vallejo; 2020. Reference Source"
}
|
[
{
"id": "165660",
"date": "19 Apr 2023",
"name": "Ioan Batrancea",
"expertise": [
"Reviewer Expertise Finance",
"Financial Analysis",
"Econometrics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments From my point of view, it is a very interesting topic and simultaneously it seems that to the best of my knowledge is the first empirical study to determine the impact of economic growth on scientific production, measured as the variation in the gross domestic product and the number of scientific publications, in Latin American and Caribbean countries. The findings of both the random effects models and the fixed effects models demonstrated the negative impact of economic growth on scientific production. This study is relevant because it is one of the first to study the impact of gross domestic product on scientific production in Latin American and Caribbean countries from a longitudinal perspective that also allows evaluating the dynamics of both variables.\nThe paper contains the following sections: Introduction, Methods, Results, Discussion and Conclusion.\nHowever, I find some recommendations:\nThere is a mistake in the key words and I propose the following order: Growth, Scientific production, Data, Analysis\n\nThe abstract must contain the main purpose of the paper, the research method used in the research and the main contributions.\n\nIt would be very useful to add in the \"Introduction\" section the purpose, objectives and hypothesis of the research. I consider that a weak point of the paper is that the authors did not show the novelty of the paper compared to other works. That is why, I consider that the introduction should specify the novelty of the paper compared to other papers published in this area.\n\nThe research is well based on science and the results are in agreement with the theoretical part. From my point of view, the paper is original and the topic addressed brings added value to the specialized literature regarding the influences of economic growth on scientific production. The paper is well written and easy to read.\n\nAt the same time, the authors are required to present Descriptive Statistics, Correlation matrix with all tests and indicators: standard deviation, Jarqe-Berra, Skewness and Kurtosis interpretation, Jarqe-Berra with probabilities analysis, etc.\n\nIt is important to present the VIF test on multicollinearity between independent variables. Heteroskedasticity and Endogeneity tests are also important in this study. All these aspects that are not found in the paper represent weaknesses of the research.\n\nAt the same time, I consider that the conclusions part of the work should be expanded with policy implications.\n\nI think that the literature needs to be improved with other works, referred to economic growth. I considered that the works cited in this paper are few and therefore the authors should expand the list of references. That is why I recommend the authors to refer to other recent works indexed in Web of Science. I suggest the authors consider the following articles, all are based on a panel econometric analysis and we have asked the authors to extend the paper with such an analysis. I suggest that the authors cite papers published in Web of Science Journals, such as:\nBatrancea L.M. (2021) An Econometric Approach on Performance, Assets, and Liabilities in a Sample of Banks from Europe, Israel, United States of America, and Canada. Mathematics, 9(24):3178. https://doi.org/10.3390/math9243178. Batrancea, L.; Rathnaswamy, M.M.; Batrancea, I. A Panel Data Analysis of Economic Growth Determinants in 34 African Countries. J. Risk Financial Manag. 2021, 14, 260. https://doi.org/10.3390/jrfm14060260 Batrancea, L.M., Rathnaswamy, M.M., Rus, MI. et al. Determinants of Economic Growth for the Last Half of Century: A Panel Data Analysis on 50 Countries. J Knowl Econ (2022). https://doi.org/10.1007/s13132-022-00944-9 Batrancea, L.M. Determinants of Economic Growth across the European Union: A Panel Data Analysis on Small and Medium Enterprises. Sustainability 2022, 14, 4797. https://doi.org/10.3390/su14084797 Batrancea, L.M.; Balcı, M.A.; Chermezan, L.; AkgÃŒller, Ã.; Masca, E.S.; Gaban, L. Sources of SMEs Financing and Their Impact on Economic Growth across the European Union: Insights from a Panel Data Study Spanning Sixteen Years. Sustainability 2022, 14, 15318. https://doi.org/10.3390/su142215318 Batrancea, L.M.; Balcı, M.A.; AkgÃŒller, Ã.; Gaban, L. What Drives Economic Growth across European Countries? A Multimodal Approach. Mathematics 2022, 10, 3660. https://doi.org/10.3390/math10193660 Shen, C.; Zhao, X. How does income inequality affects economic growth at different income levels? Econ. Res.-Ekon. Istraz. 2022, 1â22. https://doi.org/10.1080/1331677X.2022.2080742 Brueckner, M. Infrastructure and economic growth. J. Risk. Financ. Manag. 2021, 14, 543. https://doi.org/10.3390/jrfm14110543\n\nThe conclusions at the end of the paper should be expanded showing the economic policy implications of the research results.\nIn conclusion, the article should be improve. It should also be enhanced with a review of the literature adequate to the subject and a broader interpretation and commentary of the research results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "175528",
"date": "04 Jul 2023",
"name": "Lorenzo Zirulia",
"expertise": [
"Reviewer Expertise Economics of science and innovation"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper \"Impact of economic growth on scientific production in Latin America and the Caribbean based on panel data analysis\" investigates the empirical relationship between scientific publications and GDP growth rates. It finds no impact of economic growth on scientific production using robust methods. The theme is important, and overall the paper is well written. However, I have two main concerns I can list as follows.\n\nConceptually, I cannot see in clear way the mechanism through which economic growth should impact publications. There is some reference to the role of political stability, but vague. I would like to see an empirical model stating explicitly a knowledge production function, whose list of arguments would surely include R&D expenditures as primary input and other mediators. However, what would be the role of GDP growth? it is unclear to me. As it is now, GDP is apparently capturing a bunch of explanatory variables.\n\nI am also in trouble with the assumption of a contemporaneous relationship between the X and the Y. If one buys the idea that GDP growth is in fact mainly capturing R&D growth, then it would natural to assume a certain lag between R&D as input and publications as output. The non significance can be related also this issue.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "175523",
"date": "07 Jul 2023",
"name": "Paul Owusu Takyi",
"expertise": [
"Reviewer Expertise Development finance",
"macroeconomics",
"applied econometrics",
"monetary economics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments\nThe article studies the impact of economic growth on scientific production in Latin America and the Caribbean based on panel data analysis. In general, the topic and premise of the study is interesting and timely. My main concerns related to the methodology and empirical analysis. See detailed comment below.\nSpecific comments\nWhat is the overall economic growth in the region under study? What are the scientific production levels in the region? Some correlation analysis between these two variables need to be provided in the introduction section to create a research problem for the study.\n\nThe authors failed to discuss the theoretical model or theory that underpins their study. I that suggest the authors discuss the theoretical model that gave rise to the empirical model they estimated.\n\nThey employed a static panel model in their study but failed to provide a justification for it as well as the advantages of such a model over dynamic panel model.\n\nThe natural log of GDP does not directly measure economic growth as the authors use in their study. Rather, it is the log differences. I suggest that the author use the later to measure economic growth in their estimation\n\nHaving only one explanatory variable in the model used is not standard. The authors need to control for other factors could affect scientific production. For example, the level of education, expenditures on R&D etc.\n\nThe output from scientific production identifies new technology and provides policy recommendations for the governments. All of these feed into the production capacity of a country which could affect overall economic growth. Thus, there is a possible reverse causality between economic growth and scientific production leading to a potential endogeneity problem. How did they authors deal with this problem? I suggest that the authors explore other models that handles this problem (e.g. GMM, panel fixed effect with instrumental variables etc).\n\nThe equations in the text need to be numbered.\n\nThe results in Table 2 indicate that lnGDP significantly reduces scientific production. What are the possible explanations for this finding?\n\nIn the discussion section, the authors need to discuss the possible channels through which economic growth affect scientific production.\n\nThe conclusion section lacks policy implications from the results. This section needs to be strengthened.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "178689",
"date": "07 Sep 2023",
"name": "Sérgio Queiroz",
"expertise": [
"Reviewer Expertise Science and technology policy",
"economics of technology and innovation",
"technological learning processes",
"knowledge-intensive entrepreneurship",
"innovation and entrepreneurship ecosystems."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article intends to evaluate the impact of GDP growth on the number of scientific publications in 18 countries of Latin America and the Caribbean during the period 2000-2018. The study fails to show an effect of economic growth on scientific production.\nThere are a number of problems with the study, but probably the most serious one relates to its design. The paper initially states the importance of science, technology and innovation to economic development. It clearly acknowledges the role of investment in research and development to promote competitiveness and strengthen economic growth, which is known for quite some time. The literature shows since the beginning of the 20th century with Schumpeter how technological development (not scientific development, although a relationship between science and technology can be established) fosters economic growth. And since the increase of GDP is an important objective for any government, the promotion of technological development, including investment in research, human capital etc, also became an important goal. Nevertheless, the study intends to explore the inverse causation, from economic growth to science production. It is not clear what contribution this brings to our current knowledge. The results of the study are meager, but even if it had shown a strong influence of GDP growth on scientific production, what type of conclusion could be drawn from it?\nSecondly, the aggregation of 18 very different countries is not justified. Most probably the factors affecting scientific publication in large countries with significant scientific production like Brazil, Mexico, Colombia and Argentina differ considerably from what happens in countries such as Haiti or Nicaragua. It seems that the only reason to mix such a heterogeneous group of countries is to allow the application of the statistics tests selected for the study.\nOther problems could be mentioned, for instance, the number of citations from very specialized fields as representative of science production, such as Ozsoy and Demir (2018), or even journals such as âLung Cancerâ or \"Anaesthesist\" that appear in the reference list but not in the paper. However, the design problem of the study is important enough to compromise the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-242
|
https://f1000research.com/articles/12-241/v1
|
06 Mar 23
|
{
"type": "Research Article",
"title": "Evaluation of nitrate concentrations in groundwater of LlÃo and San Pablo, Guano â EC",
"authors": [
"Benito Mendoza",
"Sandra Gabriela Barrazueta Rojas",
"MarÃa Fernanda Rivera Castillo",
"Mayra Alejandra Pacheco Cunduri",
"Daniela Brito",
"Sandra Gabriela Barrazueta Rojas",
"MarÃa Fernanda Rivera Castillo",
"Mayra Alejandra Pacheco Cunduri",
"Daniela Brito"
],
"abstract": "This paper analyzes the groundwater of the Chambo aquifer in the LlÃo and San Pablo sector, with emphasis on the temporary alternation of nitrate concentration, identifying the anthropogenic activities that may influence this area. This from the cartographic definition of the areas of begging influence the crossing of geological, hydraulic and anthropic activities. The temporality of the geochemical data is 5 years (2016-2020), these results show that water quality meets the requirements of the Ecuadorian national standard, and that the main composition of water is bicarbonated type of calcium-magnesium. Even if the groundwater in this sector meets the standard, variation in nitrate concentration is evident. This type of concentration is presented by the effect of agricultural activities on the surface of the soil, where nitrates occur naturally due to the nitrogen cycle, and that are dragged into the water table by infiltration when the rainy season occurs. In addition, it was found that the type of rock found underground cannot naturally generate nitrates.",
"keywords": [
"Geology",
"Stratigraphy",
"Nitrates",
"Geochemistry",
"Chambo aquifer"
],
"content": "Introduction\n\nGroundwater runs through pores and fissures of rocks that are below the ground (Su et al., 2020), rain is the main source of food for this resource, whose particularity is that, being below the earthâs surface does not evaporate (Zárate et al., 2021). In this sense, the impact of groundwater is considered as a safe source of human consumption, and also its treatment is economical (Adimalla & Qian, 2019). In this context, 2.5 billion people meet their water consumption needs through the use of groundwater, the percentages of use of this type of water are considered to be 42 %, 36 % and 27 % for agricultural, domestic and industrial purposes respectively (Sajjad et al., 2022).\n\nIn this context, it is important to know that not all groundwater is renewable, although in many parts of the world, humans use the resource in an accelerated manner without taking into account the time it takes to replenish this resource (Agudelo Moreno et al., 2020). On a global scale, population expansion presents itself as a latent threat to aquifers, due to the development of human settlements, industries, food demand, increasing agricultural activities and the development of mining industries (Anomohanran, 2015; Sajjad et al., 2022; Smith et al., 2020; Uddin et al., 2021).\n\nOn the other hand, groundwater quality is commonly associated with chemical concentrations of different types, so the presence of nitrates, heavy metals and pathogens represent some kind of contamination in the water (Tafoya-Hernández et al., 2022). In this sense, agrochemicals bring concentrations of nutrients to the soil, these substances dissolve when water is infiltrated, and they will hit the aquifers, the concentration will depend on fluctuations in the water level, seasonality and uneven terrain (Gutiérrez et al., 2021; Navarro et al., 2021). In Latin America there are examples such as the agricultural region of Mato Grosso, Brazil, where soybean, cotton and corn crops are grown, here studies were made in the drainage area of the São Lourenço river, finding pesticides and other compounds such as nitrates in rain and groundwater samples (Casara et al., 2012). In Ecuador, studies have been made of the impact that nitrates could have on groundwater, but this in low basins such as the Daule River in the province of Guayas (Ribeiro et al., 2017). In this sense, Ecuador has legislation in favor of the prevention and conservation of underground water resources, but, even when these laws exist, principles of protection and conservation are not taken into account (Baque et al., 2016; Sandoval & GÃŒnther, 2013). Most of Ecuadorâs efforts are in the study of surface waters (Carrera-Oña et al., 2020; David et al., 2015; Guananga et al., 2022; VillamarÃn et al., 2014), leaving a significant gap in the study of groundwater in the Andean areas.\n\nOn the other hand, the Chambo aquifer located in the central area of Ecuador, provided 600 l/s of water for human consumption to the populations of Riobamba and Guano, most of the exploitation wells are in the sector of LlÃo and San Pablo, This area is characterized by being in the lower part of the slopes of the volcano Chimborazo, all this sector is of agricultural production characteristics, since there are aplias areas of crops and livestock (Chidichimo et al., 2018).\n\nIn this context, this paper analyzes the possible sources of nitrate pollution in the groundwater of the LlÃo and San Pablo sector. This using water analysis data for the period 2016 to 2020.\n\nGroundwater therefore represents an enormous resource that can only be managed if the behaviour of the aquifer with respect to the pollutants to which it is exposed is known, Therefore, this work analyzes nitrate concentrations in groundwater in the LlÃo and San Pablo sector in the period 2016-2020, to identify whether or not there is pollution with respect to Ecuadorian legislation.\n\n\nMethods\n\nThe work was carried out in the volcano-sedimentary basin of the Chambo River, in the province of Chimborazo, in the so-called Chambo aquifer (Figure 1).\n\nTo characterize the study areas, a cross-reference of bibliographic information was made, in addition to cartographic information, allowing us to know the areas of possible collection and also the flow of groundwater and the recharge zones of this (Chidichimo et al., 2018; Mendoza, 2015; Procel, 2018). Once this area was identified, the geological and stratigraphic description was made, determining the type of structure and rocks found in the subsoil of the aquifer, allowing to relate the composition of the rocks with the presence of nitrates in the water of LlÃo and San Pablo. In addition, the results obtained in the study of the Guano and Chibunga rivers (Mendoza et al., 2021), which determined the field and infiltration capacity, were used as a complementary part to understand the movement of water, to relate the presence of nitrate concentration in the infiltration process.\n\nTo define the anthropic areas and activities, the surrounding area was taken into account, indicating which are the direct and most intense anthropic activities that can influence the study area. These anthropic areas were determined by cartography, this information was used from several works carried out in the area of influence (Chidichimo et al., 2018; Mendoza, 2015; Mendoza et al., 2021; Procel, 2018).\n\nThe water analysis results obtained from 2016 to 2020 were used to determine the behaviour of nitrate concentrations in water, taken from the analysis reports provided by the Environmental Services Laboratory of the National University of Chimborazo. In addition, laboratory analysis results of EP-EMAPAR from 2020 were used (Mendoza et al., 2023).\n\nThe nitrate determination of groundwater by the Environmental Services Laboratory was carried out in accordance with the Standardized Methods for Examining Wastewater and Water (Bluett et al., 2022; Bodrud-Doza et al., 2019; Daghara et al., 2019; Rice et al., 2017), and developed according to the following procedure (Hach Company, 2000): 1) Shake the water sample for 1 minute to homogenize, 2) take an aliquot of 25mL of the sample to be analyzed in the glass cell (code HACH-20950-00), 3) add the contents of a bag of nitrate reagent powder NitraVer 5 (code HACH-14034-99) to the cell, cover, 4) shake the cell with the sample for one minute, 5) after shaking the sample let stand for five minutes, 6) take an aliquot of 25mL of deionised water in the glass cell (code HACH-20950-00) for use as a target, 7) place the analysis code for Nitrates N03-N on the HACH UV/VIS DR5000 instrument, leaving the wavelength of the equipment at 500nm, 7) place the cell with deionised water on the computer and press ZERO, 8) place the cell with the sample on the computer and press READ, 9) record the data.\n\nOnce the geological strata where the sampling points of the slope and the wells are located were identified, these were classified by the geological and stratigraphic composition. Afterwards, the results of water analysis of LlÃo and San Pablo were interpreted according to their geochemical composition, to determine the type of water and its origin, this through the diagram of Piper (Albo & Blarasin, 2014) and geological description made by Salguero (2017) and Procel (2018). The anthropic activities present in the area of influence were also identified and the nitrate concentration data were obtained temporarily. At the end, the comparison was made with respect to the norms that show admissible levels of nitrates shown in Table 1, for water for human consumption that must meet quality standards to avoid risks of diseases (Baque et al., 2016).\n\n\nResults and discussion\n\nThe work was carried out in the volcano-sedimentary basin of the river Chambo, in the inter-Andean depression, in this area the mesozoic basement is covered by sedimentary sequences, pleocene and pleistocene, constituted in the region by the Jurassic rocks of the Alao Paute Unit, Guamote, Daldal River, San Pablo de Sali and the Cretaceous rocks of the Peltetec Unit and Yunguilla Formation (Chidichimo et al., 2018; Mendoza, 2015; Procel, 2018). The units that form the base of the lower basin of the Chambo River, are located in the Cordillera Oriental, with respect to the study area that is the sector of LlÃo and San Pablo.\n\nAs described by Procel (2018) in the area there are three aquifers, of the multilayer type, called LlÃo-Guano, Riobamba and YaruquÃes. The LlÃo-Guano Aquifer is located near the Chimborazo and Igualata volcanoes, this aquifer is of the groundwater type (free), with double porosity, average width of 200 m, associated with the volcanogenic deposits of the Chimborazo (upper) and Cizarán (lower) formations. The average flow rate is 0.27 m3/s, of which 0.039 m3/s correspond to the flow rates of the wells located in LlÃo and Guano. The rest, 0.23 m3/s, corresponds to the source San Pablo, located approximately one km southwest of the wells of LlÃo. These wells and the spring are the source of water for public supply in the city of Riobamba and Guano. The hydraulic conductivity is in the order of 6.1x10-6 m/s and the average water table level is 65 m, in the wells located in the Chimborazo Formation, and 140 m in the drilled in the Cizarán Formation. The predominant direction of water flow is from northwest to northeast (from Chimborazo to the Chambo River) and from north to south (from Igualata to the Guano River) (Procel, 2018).\n\nOn the other hand, the stratigraphy of the area of LlÃo and San Pablo, shows that the area of the wells mostly consists of permeable areas, since it contains lithological profiles of sand, thick gravels and stones. In addition, it has semipermeable areas composed of gravels, clayey sandstones and quartzites, healthy schists and altered granite. Waterproof areas are presented in the form of healthy lavas and granites. The San Pablo slope presents permeable areas with the presence of medium and thick sand, pebbles, coarse gravel and sandy brown clay, in addition to waterproof areas that represent sands and gravels, clay sands and quartzites (Mayorga, 2020). In this context it was identified that the main stratigraphy of the study area presents fine, medium and thick sands, coarse gravels, angular gravel, ridges, clay sandstones, sandy brown clay, quartzes, healthy schists, altered granite, healthy granites, coarse granite, medium granites, lavas (Mayorga, 2020; Procel, 2018)\n\nTo describe the field capacity and infiltration, the area of influence was first identified (Figure 2), crossing information between: type and use of the soil, vegetation cover, geology and geomorphology of the area. Determining according to the geomorphology there is incidence of two sources: from the volcano Chimborazo and from the volcano Igualata.\n\nIdentified two areas that do not allow infiltration such as bodies of water and snow. In addition, three types of areas in the upper parts of both volcanoes: paramo up to 4000 m.s.n.m, cultivated grass and short-cycle crops, this in the range of 4000 to 3200 m.s.n.m. In this context it is shown that there is great capacity and infiltration in this area (Table 2) since we have an average of 112.04 mm/h (Mendoza et al., 2021). The hydrogeochemical classification of groundwater considered three samples from LlÃo #5 well and San Pablo spring collected between December 2017 and August 2019 (Table 3).\n\nThe contrast of information from the results of the physical-chemical and chemical analyses in samples from the well and the spring indicated the occurrence of a predominant type of water with the presence of calcium-magnesium bicarbonates in LlÃo and San Pablo (Figure 3).\n\nTo analyze the variation in nitrate concentration, the pumped water collection system of EP-EMAPAR and the San Pablo spring were considered (Figure 4). As seen in the figure the wells are located in an area with anthropic influence, since around the two sources there are areas of cultivation and pasture.\n\nIn this context, from January 2016 to December 2020, monthly sampling of water from wells 1, 2, 3, 4, 5, 6 and 7 of LlÃo and the San Pablo spring was carried out (Table 4), these analyses were carried out in the Environmental Services Laboratory, EP-EMAPAR Laboratory. As shown in Figure 5, it is evident that there is variation in nitrate concentration, in addition the values do not exceed the maximum permissible limit of standards: INEN NTE 1108 (INEN, 2020), Guidelines for the quality of water for human consumption (WHO, 2018), Ministerial Agreement No 097 (MAE, 2016).\n\nAlthough there are no values that affect the health of people who consume water from this pumping system, differences in nitrate concentration were found each year (Figure 6), by 2016 there were a minimum of 1,74 mg/L and a maximum of 18,12 mg/L, for 2017 the minimum is 1,90 mg/L and the maximum is 21.96 mg/L, for 2018 the minimum is 1,21 and the maximum of 17.68 mg/L, in 2019 the minimum is 1,00 mg/L and the maximum of 25,90 mg/L. The average annual value is 7,4 mg/L among all the years of study, the standard deviation of the obtained results is 3,75 mg/L, this implies a very high value in relation to errors in the measurement in laboratory or in the collection of samples.\n\nTo establish the natural concentration of nitrates in the study area, we describe the microscopic analysis of the lithology of the influence zone described in the âGeological-structural levation of the area covered by the topographic sheet of Guano scale 1:50,000. Chimborazo Provinceâ (Salguero, 2017). First, lava flows are observed that have similar minerology, with serial dimensional porphyric texture and are composed of crystals of:\n\n1. âPlagioclase (30%) idiomorphic to subaiomorphic, with Carlsbad type maclas, with structures in gulfs and reaction edges, granulometric variable, in sizes from 0.6 to 2.5 mm and with a composition of andesin and oligoclase (for smaller crystals)â.\n\n2. âClinopyroxines (8%) and allotriomorphic orthopyroxenes (7%) at high morphic, with reaction edges and sizes < 1mmâ.\n\n3. âThe matrix (52%) varies according to distance and is composed of plagioclase microliths, pyroxenes, volcanic glass and iron oxides (< 3%)â.\n\nThese andesitic lava structures are immediately arranged over the avalanche deposit of debris from the eruption of the Chimborazo volcano and contain only thin layers of regolith and soil. These remains of andesitic lava were deposited and filled with ravines and ancient valleys, formed inside the deposit of the avalanche of debris from the eruption of the Chimborazo volcano (Procel, 2018) (Salguero, 2017).\n\nAs shown in the description of Salguero (2017), the type of rocks and their composition shows mostly rocks composed of silica, calcium, sodium, aluminum, magnesium, manganese, lithium, iron, iron oxides. In addition, the geochemistry of the water samples presented in paragraph 4.6 also shows that the water present in LlÃo well 5 and San Pablo spring have an abundance of calcium bicarbonates-magnesium, so naturally Nitrates could not originate in the geological complex that was studied, passing the process of nitrogen formation by the presence of organic, atmospheric and anthropic components.\n\nFrom the above it is evident that the major influence towards the area of LlÃo and San Pablo is given by the topography of the area. In this case, both the upper part of the Chimborazo volcano and that of the Igualata volcano influence the flow of surface and underground water, and therefore the infiltration of water in this area.\n\nFigure 2 shows paramo areas in each area of the volcanoes which causes water retention and great infiltration power to the aquifer in this area, we could say that these two areas are the water recharge areas for this groundwater system. In addition, it is observed in the intermediate part before the upper limit of the aquifer of the same Figure 2, areas with cultivated grass and crops of short cycle. These areas have as main property the presence of organic matter in the upper part of the soil (Mendoza et al., 2020). Thus, the aforementioned can be said that the nitrates present in the surface layers of the soil in the study area is due to the effect of the nitrogen cycle (Holloway & Dahlgren, 2002). Therefore, the infiltration capacity of the study area allows the nitrogen compounds to move into the aquifer. In addition, the rainy season in the area should be taken into account (Chidichimo et al., 2018), since from March to May there is a greater amount of precipitation so it influences the washing of the soil and the drag of all types of compounds into the water table. All this group of details present in the area are those that probably affect the groundwater and its concentration of nitrates of LlÃo and San Pablo.\n\n\nConclusions\n\nGeology, stratigraphy, lithology in conjunction with the study of field capacity and infiltration allow to know the area of physical influence of LlÃo and San Pablo, as shown in the document volcanic structures are observed, lava flows and avalanche deposits that drastically influence the infiltration and movement of surface and groundwater. In determining the zone of influence, it was determined that the upper parts where the moors are present are the areas where the aquifer is recharged as they receive water from the precipitation and in addition to the melting of the Chimborazo volcano.\n\nThe geochemical study allowed to know the characteristics of the water obtained in the sector of LlÃo and San Pablo, according to the reported analyses and its interpretation in relation to the ions and cations present in this was determined to have water characteristics with bicarbonates of calcium-magnesium mostly.\n\nThe variety of nitrates in the groundwater of LlÃo and Sú Paulo shows that the concentrations of nitrates are lower than suggested by water quality standards such as INEN NTE 1108, Ministerial Agreement 097-A and those described by the WHO, therefore, in this sense the water maintains good quality with regard to nitrate concentrations. However, there have been strong variations over the period under study, reaching a figure of more than 20 mg/L in some months, which, although still within the standards, is worrying as nitrate levels rise at certain times of the year.\n\nTherefore, according to what was studied in this work, it can be noted that the presence of nitrate concentration is mainly due to biological and geochemical effects that occur in the surface of the study area, mainly in areas where pastures are cultivated as there is presence of animals that contribute a good amount of nitrogen compounds such as urea. In addition, it is evident the presence of areas with short-cycle crops, in which natural and artificial fertilizers with nitrogen composition are used. In this context, soil studies are necessary to corroborate the information on the presence of nitrates. To all this, the rainfall regime in the Ecuadorian highlands is added and as shown the good infiltration capacity of the area, make the nitrogen cycle produce a good portion of nitrates that go to the groundwater.\n\nThe water that comes from LlÃo and San Pablo is of good quality according to the reports used in this research work, but it must be taken into account that the variation of nitrates found in it implies that anthropic pressure exists in the area of origin of this, so it should be considered to keep free of activities at least the area of moors in the recharge area to preserve this resource that is essential for the population of Riobamba and Guano.",
"appendix": "Data availability\n\nZenodo. Evaluation of nitrate concentrations in groundwater of LlÃo and San Pablo, Guano â EC. DOI: 10.5281/zenodo.7559363 (Mendoza et al., 2023).\n\nThis project contains the following data:\n\n- This paper analyzes the groundwater of the Chambo aquifer in the LlÃo and San Pablo sector, with emphasis on the temporary alternation of nitrate concentration, identifying the anthropogenic activities that may influence this area. This from the cartographic definition of the areas of begging influence the crossing of geological, hydraulic and anthropic activities. The temporality of the geochemical data is 5 years (2016-2020), these results show that water quality meets the requirements of the Ecuadorian national standard, and that the main composition of water is bicarbonated type of calcium-magnesium. Even if the groundwater in this sector meets the standard, variation in nitrate concentration is evident. This type of concentration is presented by the effect of agricultural activities on the surface of the soil, where nitrates occur naturally due to the nitrogen cycle, and that are dragged into the water table by infiltration when the rainy season occurs. In addition, it was found that the type of rock found underground cannot naturally generate nitrates.\n\n\nReferences\n\nAdimalla N, Qian H: Groundwater quality evaluation using water quality index (WQI) for drinking purposes and human health risk (HHR) assessment in an agricultural region of Nanganur, south India. Ecotoxicol. Environ. Saf. 2019; 176: 153â161. PubMed Abstract | Publisher Full Text\n\nAgudelo Moreno LJ, Zuleta Lemus D d S, Lasso Rosero J, et al.: Evaluation of aquifer contamination risk in urban expansion areas as a tool for the integrated management of groundwater resources. Case: Coffee Growing Region, Colombia. Groundw. Sustain. Dev. 2020; 10: 100298. Publisher Full Text\n\nAlbo G, Blarasin M: Hydrogeochemistry and estimation of the natural nitrate background of groundwater in an agroecosystem of the pedemonte of the Comechingones mountain range. Journal of the Argentine Geological Association. 2014; 71(3): 378â392.\n\nAnomohanran O: Hydrogeophysical investigation of aquifer properties and lithological strata in Abraka, Nigeria. J. Afr. Earth Sci. 2015; 102: 247â253. Publisher Full Text\n\nBaque R, Simba L, González B, et al.: Quality of water intended for human consumption in a canton of Ecuador/Quality of water intended for human consumption in a canton of Ecuador. UNEMI SCIENCE. 2016; 9(20): 109â117. Publisher Full Text\n\nBluett S, OâCallaghan P, Paull B, et al.: Robust off-grid analyser for autonomous remote in-situ monitoring of nitrate and nitrite in water. Talanta Open. 2022; 7: 100173. Publisher Full Text\n\nBodrud-Doza M, Islam SMDU, Hasan MT, et al.: Groundwater pollution by trace metals and human health risk assessment in central west part of Bangladesh. Groundw. Sustain. Dev. 2019; 9: 100219. Publisher Full Text\n\nCarrera-Oña GE, Santiana-EspÃn CG, González-Palacios MA, et al.: Design of experiments in the analysis of nitrate concentration and pH levels in water samples from the Microcuenca del Rio Chibunga - Ecuador. Pole of Knowledge. 2020; 5(5): 439â452. Publisher Full Text\n\nCasara KP, Vecchiato AB, Lourencetti C, et al.: Environmental dynamics of pesticides in the drainage area of the São Lourenço River headwaters, Mato Grosso State, Brazil. J. Braz. Chem. Soc. 2012; 23(9): 1719â1731. Publisher Full Text\n\nChidichimo F, Mendoza BT, De Biase M, et al.: Hydrogeological modeling of the groundwater recharge feeding the Chambo aquifer, Ecuador. AIP Conference Proceedings. 2018; 2022. Publisher Full Text\n\nDaghara A, Al-Khatib IA, Al-Jabari M: Quality of Drinking Water from Springs in Palestine: West Bank as a Case Study. J. Environ. Public Health. 2019; 2019: 1â7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavid I, Carrera Villacrés V, Sonia I, et al.: Salinity, Phosphates, Nitrates and Infiltration Problems In the irrigation waters of the canton Milagro, Ecuador. UNEMI SCIENCE. 2015; 6(9): 85â95. Publisher Full Text\n\nGuananga N, Mendoza B, Guananga F, et al.: Influence of geomorphology and flow in the water quality of the Guano River, Ecuador. Novasinergia. 2022; 5(2): 174â192. 2631-2654. Publisher Full Text\n\nGutiérrez M, Alarcón-Herrera MT, Calleros-Rincón EY, et al.: Nitrate in agricultural soils. Nitrate Handbook: Environmental, Agricultural, and Health Effects. 2021; 25â43. Publisher Full Text\n\nHach Company: Manual of water analysis. Cell. 2000; 3(970). Reference Source\n\nHolloway JAM, Dahlgren RA: Nitrogen in rock: Occurrences and biogeochemical implications. Glob. Biogeochem. Cycles. 2002; 16(4): 65-1â65-17. Publisher Full Text\n\nMayorga M: Three-dimensional geophysical modelling of the Llio pumping system, EP EMAPAR. 2020, September 18. Undergraduate thesis. Reference Source\n\nMendoza B: Characterization of real aquifers using hydrogeophysical measurements. An application to the chambo aquifer (Ecuador). Tesi di Dottorato. 2015; 208. Reference Source\n\nMendoza B, Fiallos M, Iturralde S, et al.: Determination of field capacity in the Chibunga and Guano rivers micro-basins. F1000Res. 2021; 10: 172. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMendoza B, Guananga N, Melendez JR, et al.: Differences in total iron content at various altitudes of amazonian andes soil in Ecuador. F1000Res. 2020; 9: 128. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMendoza Trujillo Benito Guillermo, Brito Arteaga Daniela, Barrazueta Rojas Sandra Gabriela, Rivera Castillo MarÃa Fernanda, & Pacheco Cunduri Mayra Alejandra. Evaluation of nitrate concentrations in groundwater of LlÃo and San Pablo, Guano â EC. 2023.Publisher Full Text\n\nNavarro L, Camacho R, López JE, et al.: Assessment of the potential risk of leaching pesticides in agricultural soils: study case Tibasosa, Boyacá, Colombia. Heliyon. 2021; 7(11): e08301. PubMed Abstract | Publisher Full Text | Free Full Text\n\nProcel S: Ferramentas de gestão dos recursos hÃdricos: estudo de caso na bacia do baixo Rio Chambo (Equador) [Biblioteca Digital de Teses e Dissertações da Universidade de São Paulo].2018. Publisher Full Text\n\nRibeiro L, Pindo JC, Dominguez-Granda L: Assessment of groundwater vulnerability in the Daule aquifer, Ecuador, using the susceptibility index method. Sci. Total Environ. 2017; 574: 1674â1683. PubMed Abstract | Publisher Full Text\n\nRice E, Baird R, Eaton A: Standard Methods for the Examination of Water and Wastewater ed-23rd. American Public Health Association (APHA), American Water Works Association (AWWA) and Water Environment Federation (WEF); 2017. Reference Source\n\nSajjad MM, Wang J, Abbas H, et al.: Impact of Climate and Land-Use Change on Groundwater Resources, Study of Faisalabad District, Pakistan. Atmos. 2022; 13(7): 1097. Publisher Full Text\n\nSalguero D: Geological-structural survey of the area covered by the Guano Topographic Sheet Scale 1:50000, Chimborazo Province. 2017, December 13. Undergraduate thesis. Reference Source\n\nSandoval A, GÃŒnther M: Community water management in Mexico and Ecuador: Other approaches to sustainability - Dialnet. Ra Ximhai, Scientific Journal of Society, Culture and Sustainable Development. 2013; 9: 165â179. Reference Source\n\nSmith DNI, Ortega-Camacho D, Acosta-González G, et al.: A multi-approach assessment of land use effects on groundwater quality in a karstic aquifer. Heliyon. 2020; 6(5): e03970. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSu YS, Ni CF, Li WC, et al.: Applying deep learning algorithms to enhance simulations of large-scale groundwater flow in IoTs. Appl. Soft Comput. 2020; 92: 106298. Publisher Full Text\n\nTafoya-Hernández A, Talavera-Mendoza O, Salgado-Souto SA, et al.: Impact assessment of HgâSb wastes to urban soils and shallow groundwater in the mining region of Huitzuco, Guerrero (southern Mexico) using mineralogical, geochemical and SrâPb isotopic tools. Appl. Geochem. 2022; 138: 105213. Publisher Full Text\n\nUddin MG, Nash S, Olbert AI: A review of water quality index models and their use for assessing surface water quality. Ecol. Indic. 2021; 122: 107218. Publisher Full Text\n\nVillamarÃn C, Prat N, Rieradevall M: Physical, chemical and hydromorphological characterization of the tropical high Andean rivers of Ecuador and Peru. Lat. Am. J. Aquat. Res. 2014; 42(5): 1072â1086. Publisher Full Text\n\nZárate BA, El Hamdouni R, Fernández T: GNSS and RPAS Integration Techniques for Studying Landslide Dynamics: Application to the Areas of Victoria and Colinas Lojanas, (Loja, Ecuador). Remote Sens. 2021; 13(17): 3496. Publisher Full Text"
}
|
[
{
"id": "168048",
"date": "04 Apr 2023",
"name": "Tiziano Boschetti",
"expertise": [
"Reviewer Expertise hydrogeochemistry",
"isotope geochemistry",
"analytical geochemistry"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Mendoza et al. presents interesting data on a long-term monitoring of nitrate content in groundwater in an area of Ecuador. Surely, the work could be of interest to F1000 Research readers. However, the work needs improvement. Firstly, the English grammar and style does not seem appropriate for a scientific publication. I have tried to correct it starting from the abstract, but I have limited myself to that since I do not think that it is a reviewer's task. Therefore, I recommend a thorough stylistic and grammatical editing of the English by a proofreading service. Secondly, the methods report an \"operational\" description of the nitrate analysis method used in the research, but the authors do not mention some very important things: 1) where the method comes from - it is a modification of the Cadmium Reduction Method, 4500-NO3 E (Standard Methods, 2017, https://www.standardmethods.org/doi/abs/10.2105/SMWW.2882.089); 2) no indication is given of the precision and accuracy of the HACH method; 3) no indications are given on the sampling and sample preservation procedures used.\nConcerning the results and discussion section: table 3 shows other parameters in addition to nitrates (main constituents, manganese...), but the analytical methods for these parameters are not described in the methods section (moreover, pH and temperature are missing). Additionally, the Piper plot is not correct (nitrate appear in the anion corner instead of chloride).\nIn the drinking water limits, Table 1, the nitrate limits expressed as mg/l as N (from EPA) are confused and compared with others that have values expressed in mg/l as NO3 (see detailed comments below).\nFinally, the graph that could be defined as the most important for variability (Fig. 5) does not show monthly rainfall, which could be helpful for interpretation. Moreover, since the local rainfall data could be very important, there is no description of the local climate.\nTherefore, the work should be heavily revised before being accepted for indexing. See the detailed comments below. Hope this helps.\nDetailed comments:\nAbstract: italic: rewritten âThis latter data have been acquired by the cartographic definition of the areas affected by the crossing of geological, hydraulic and anthropic activities.â ââŠEcuadorian national water quality standard, and that the main composition of water is bicarbonate-Ca(Mg)âs typeâ ââŠConsidering that local unaltered rocks do not contain relevant amount of nitrogen, such kind of variation is mainly due to the effect of agricultural activities on the soil surface. There, nitrates occur naturally due to the nitrogen cycle, then dragged into the water table by infiltration when the rainy season occurs.â\nFigure 1: Comment: I suggest to add an inset map of the Northern South America with the Chimborazo are within Ecuador (e.g.: https://reliefweb.int/map/costa-rica/northern-south-america).\nMethods section: I suggest to add the sampling procedure and water conservation till to analysis. In particular: i) please explain if the water necessitate of filtration (colored water due suspended matter could generate intereference on that method). ii) please explain if the water was stored in dark and at 4°C before analysis. iii) please describe the time elapsed from sampling and analysis: it was compliant for nitrate conservation? Check this on the cited Standard Methods. Finally, I suggest to substitute this sentence: âThe nitrate determination of groundwater by the Environmental Services Laboratory was carried out in accordance with the Standardized Methods for Examining Wastewater and Water (Bluett et al., 2022; Bodrud-Doza et al., 2019; Daghara et al., 2019; Rice et al., 2017), and developed according to the following procedure (Hach Company, 2000):âŠâ With the following: âThe nitrate determination of groundwater is an adaptation of the so-called Cadmium Reduction Method, 4500-NO3 E (Baird et al., 2017), according to Method 8039 (Hach Company, 2000):âŠâ\nI suggest also to describe what is the precision and accuracy of the method, along with the limit of detection, the limit of quantification, and the maximum concentration accepted by the method (i.e., mix-max concentration range in mg/L as NO3).\nResults and discussion: Table 1: Please note that (ATSDR, 2017): âThe EPA lists maximum contaminant levels (MCL) and maximum contaminant level goals (MCLG) of 10 mg/L for nitrate (as nitrate-nitrogen; ~44 mg nitrate/L) and 1 mg/L for nitrite (as nitrite nitrogen; ~3.3 mg nitrite/L) in the 2012 Edition of the Drinking Water Standards and Health Advisories (EPA 2012)â. However, that concentration has been confirmed in the table published more recently (EPA, 2018). Therefore, in the Table 3 concentration should be corrected for EPA = 44 mg/l as NO3-. I also suggest to add the reference for WHOâs guideline value (WHO 2016).\nAt page 6 of 15, the authors say: âThe average annual value is 7,4 mg/L among all the years of study, the standard deviation of the obtained results is 3,75 mg/L, this implies a very high value in relation to errors in the measurement in laboratory or in the collection of samples.â I partially agree with this. Surely, a coefficient of variation higher 50%, calculated as C.V. = (standard deviation/mean)x100 is surely high. Rather, I suggest to show that value per year in each well and spring in Table 4. Say that, it is not so clear from the text whether the authors impute this variability due to the âanalytical errorâ, also because that error is not mentioned in the methods (see the comment above). However, in my opinion, this could rather be due to soil run-off effects or water table recharge as a consequence of rainfall. Therefore, it could be useful to add the rainfall per month in mm in Fig. 5, for example using a two y-axes diagram, and observe if there are any similar (or more likely inverse) trends to that of nitrates. That would be more in line with the conclusions.\nTable 3: It seems that also other parameters including main dissolved cations and anions have been determined, but the analytical methods were not described. Moreover, why was only well 5 analyzed? Please also note that zero concentration does not exist in analytical chemistry. Therefore, the authors should write.\nFigure 3 (Piper Diagram): the diagram seems to be wrong because in the anion ternary diagram there are nitrate instead of chloride. Moreover, main chemistry of the water is Mg(Ca)-bicarbonate and not Ca(Mg)-bicarbonate.\nFigure 5: what is the meaning of the two red squares at the start and the end of the diagram?\nAt page 11 of 15 the authors say: âFigure 2 shows paramo areas in each area of the volcanoesâŠâ. I warmly suggest to shift there the Fig. 2 and to renumber the figures' order.\nFigure 6: I suggest to modify the caption as âBox plot of nitrate concentrationâŠâ.\n\nReferences:\nATSDR (2017) Toxicological profile for nitrate and nitrite. U.S. Department of healt and human services, Public Health Service, Agency for Toxic Substances and Disease Registry. https://www.atsdr.cdc.gov/toxprofiles/tp204.pdf\n\nBaird, R.B., Eaton, A.D. and Rice, E.W., Eds. (2017) Standard Methods for the Examination of Water and Wastewater. 23rd Edition, American Public Health Association, American Water Works Association, Water Environment Federation, Washington D.C.\n\nEPA (2012). Drinking water standards and health advisories. Washington, DC: U.S. Environmental\n\nProtection Agency, Office of Water. EPA822S12001. http://water.epa.gov/drink/standards/hascience.cfm. January 08, 2014.\n\nEPA (2018) EPA Drinking Water Standards and Health Advisories tables https://www.epa.gov/system/files/documents/2022-01/dwtable2018.pdf\n\nWHO (2016) Nitrate and Nitrite in Drinking-water. Background document for development of WHO Guidelines for Drinking-water Quality. WHO/SDE/WSH/7.01/16/Rev/1. https://cdn.who.int/media/docs/default-source/wash-documents/wash-chemicals/nitrate-nitrite-background-jan17.pdf?sfvrsn=1c1e1502_4\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "274288",
"date": "29 May 2024",
"name": "Pantelis Sidiropoulos",
"expertise": [
"Reviewer Expertise Simulation and Optimization of Groundwater Resources",
"Quantity and Quality of Groundwater"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Mendoza et al. analyzes the groundwater quality of an aquifer in Ecuador for 5 years (2016-2020) focusing on nitrate concentrations. Although the main objectives of the submitted manuscript are in line with the Aims and Scope of the journal, it has many flaws â which I present below\n\nGeneral Comments Line numbering is needed to indicate the remarks. Extensive editing of the English language is required, as the manuscript contains many spelling, syntax, and grammatical errors. Furthermore, many phrases are quite often repeated such as âOn the other handâ, and âIn this contextâ. Furthermore, the way/method/process that identifies the anthropogenic activities which influence the groundwater quality lacks a scientific base and this is because the manuscript does not contain any information on land uses (e.g. a map). The structure of the manuscript has flaws. Please see my comments below. The Introduction chapter is very short. I see no literature review. The introduction of the paper should be enriched so that: i) the cited literature is cached in such a way that the relevance to the current research is clearer, and the need for the current research is well-founded, and ii) the contribution of the work to be better highlighted. The study area description is in the Results and Discussion chapter, which is not correct. It should be moved to the Methods chapter and should be enriched with more information regarding the climate, hydrology, topography, land uses, etc. The âMethodsâ Chapter is too short. It should be enriched with more information regarding: 1) the origin of the method used, 2) the location of the sampling (here and not in the chapter on the results), 3) the parameters that have been determined, and the reason for their selection (here and not in the chapter of the results), (4) the sampling and sample preservation procedures used. Authors follow an unorthodox way of presenting a method, a process, or a case (e.g. the study area) = they believe that is enough to just write down one or more references that they have dealt with a method, a process, or a case without presenting them even though very briefly. For example, in the last paragraph of the Methods chapter is written ââŠ.to determine the type of water and its origin, this through the diagram of Piper (Albo&Blarasin, 2014) and geological description made by Salguero (2017) and Procel (2018)â which is this geological description? There are tables and figures in the manuscript that have not been properly and analytically discussed e.g. Figure 5.\nSpecific Comments The last paragraph of the Introduction chapter is a repetition of the previous one. The authors should dedicate one paragraph describing very analytically the target of the study. Figure 1: It is âLegendâ and not âLeyendâ. A general map of Ecuador indicating the Chimborazo province and the aquifer is preferable instead of the map of the Chimborazo province (Map at the right side of the figure). It is difficult to read the main map: What do the different colors of the zones/polygons indicate in the map? Is it the geological structure? Legend, please. What the black dots are? The title âWells and springsâ should not be located on the map but in the Legend. The caption of the figure should be enriched with the description of the data shown. Table 1 cannot be in the Results and Discussion chapter. It should be moved to the Methods chapter. The references of the norms should be presented. In the Results chapter there is confusion regarding the use of commas and dots for the decimals. Please use the dot. Table 3. Why only the well 5 is chosen? The paragraph presenting the three aquifers should be accompanied by a map. Not forgetting to move the description of the study area in the Methods chapter. Figure 3. Why in the anion ternary diagram there are nitrate instead of chloride? Figure 4 is too zoomed, and the reader cannot understand the location of the wells and the spring in the aquifer area. Figure 5. A legend should be added to the graph indicating what each line represents. Figure 6 should be located in the Results chapter\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-241
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https://f1000research.com/articles/10-36/v1
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19 Jan 21
|
{
"type": "Research Article",
"title": "The disconnect between researcher ambitions and reality in achieving impact in the Earth & Environmental Sciences â narrowing the gap",
"authors": [
"Andrew Kelly",
"Victoria Gardner",
"Anna Gilbert",
"Victoria Gardner",
"Anna Gilbert"
],
"abstract": "Background: There is an increasing desire for research to provide solutions to the grand challenges facing our global society, such as those expressed in the UN SDGs (âreal-world impactâ). Herein, we consider whether the frameworks that underpin the research endeavour are appropriately oriented to support these aspirations and maximize the capability of research to achieve these goals. Methods: We conducted a survey of authors who had published in >100 of our Earth & Environmental Science journals. The survey was sent to just under 60,000 authors and we received 2,695 responses (4% response rate).\n\nResults: Respondents indicated that the majority of their research in the Earth & Environmental Sciences is currently concerned with addressing urgent global needs or that this will become a priority in the future; however, the impetus seems to be altruistic researcher desire, rather than incentives or support from publishers, funders, or their institutions. Indeed, when contextualised within other forms of impact, respondents indicated that citations or downloads were more important to them than contributing to tackling real-world problems. Herein, we analyse survey feedback, suggest the presence of a misalignment between researcher ambition and current realities, and discuss the role and value of the research journal in forming new connections for their researchers, both within and without academia. Conclusions: At present, it seems that this laudable ambition of achieving real-world impact is seemingly being lost amidst the realities of being a researcher. We offer for comment a series of suggestions, with the aim of simulating discussion and collective action to tackle these challenges as a community.",
"keywords": [
"academic publishing",
"Earth Sciences",
"Environmental Sciences",
"research journals",
"research assessment",
"survey",
"Sustainable Development Goals",
"SDGs"
],
"content": "Introduction\n\nAlthough one of the fundamental tenets of the research endeavour is about exploration and curiosity, calls to increase R&D spending by nations have been allied with an expectation for research to have âimpactâ1. Impact has many forms â some are easily quantifiable, for example every dollar invested in the Human Genome Project returned $141 to the US economy2, whereas others are harder to quantify and often tie into complex interdisciplinary issues, which require long-term commitment and investment â the UNâs Sustainable Development Goals (SDGs) and the missions of Horizon Europe serving as prime examples3,4. This drive for research to solve âthe Grand Challenges of our timeâ5 has acquired increased urgency during the Covid-19 pandemic, but has long been discussed in the context of global problems, including climate change, food security, and an aging global population. Where once there was a comment that we no longer needed experts6, trust in research and researchers to provide solutions to real-world problems is currently high, with seven in ten people commenting before the Covid-19 pandemic that science benefits them7, and trust in advice from âqualified scientists and researchersâ growing even faster in the face of the pandemic8,9.\n\nHow do these aspirations manifest at the level of those carrying out research? Primarily, itâs through research-assessment mechanisms linked to institutional or grant funding, with citations being the primary currency of success or progress. The UKâs Research Excellence Framework (REF)10 exercise has previously linked to the Pathways to Impact initiative11, with contributions to the REF expected to provide impact case studies alongside their submissions that showcase their social or economic impact. It can be challenging to see how answering one research question can create a chain reaction that resonates at a much-higher level, still less how interrelated research questions contribute to solutions to these complex and interdisciplinary issues.\n\nThe role that academic publishing plays in the advancement of research is often understood to comprise validation (through the peer-review process), publication (participation in the scholarly record), curation (preservation of the work to ensure its availability in perpetuity), and dissemination (to relevant communities). However, it is becoming increasingly apparent that the value of a research journal is much broader than this, additionally fostering collaboration, network-building (both within core and adjacent fields), and career development12.\n\nTherefore, it is essential that the mechanisms and drivers that collectively influence where an author chooses to publish their research support their ability to publish in the journals that are most relevant to their work; that is, where their research is most likely to be found, read, cited, and iterated upon by those working in the same and adjacent disciplines, as well as by those working in policy-making, lobbying, or advisory capacities. However, such drivers and pressures, both personal and external, are varied and nuanced, as are our authorsâ expectations for what impact that their work might have once it has been published.\n\nIt is in this context that we undertook the 2020 Impact Assessment of Earth & Environmental Sciences Research: Author Survey. The survey was designed to achieve three main aims. To understand:\n\nwhat drives our communities to choose the topics that they research;\n\nwhat drives our communities to choose the journals that they publish in; and\n\nwhat type(s) of impact they are most looking for from their work.\n\nWe investigated what benefits publishing in our journals could impart on both the research and on the authors following publication, and we looked at to what extent global challenges, such as those expressed by the UN SDGs, were shaping researcher ambitions. This report describes the results that we obtained and considers how the frameworks that are currently in place in academic publishing, such as those around securing research funding, researcher assessment, and career progression, are shaping the desires and decisions of our researchers, either in support of, or in opposition to, these desires.\n\n\nMethods\n\nIn Spring 2020, Taylor & Francis surveyed authors from across our Earth & Environmental Sciences portfolio. The survey (see Extended data13), hosted on Alchemer (formerly SurveyGizmo), was emailed to authors using Salesforce Marketing Cloud. It was sent to just under 60,000 authors and received 2,695 responses (4% response rate).\n\nThe survey comprised 23 questions: section A (Q1 & 2) = multiple choice questions to clarify the article that the survey responses related to; section B (3 & 4) = multiple choice questions with the option of prose responses relating to the choice of journal; section C (5â10) = multiple choice questions with the option of prose responses relating to the downstream value of publishing the article for both the work and the author; section D (11â20) = largely multiple choice with the option of prose relating to the impact of the work, the motivation for undertaking the work, and the ability of the work to tackle real-world problems and influence policy change. Questions 13, 15, 18, and 20 were solely prose responses. Section E (21â23) = demographic questions.\n\nA confidentiality and privacy statement was provided on the first page of the survey, which outlined how the data would be used. Consent to participate in the survey was implied by the authors who clicked through to complete the questionnaire after reading this statement and the instructions given in the invitation email. The data are fully anonymized and no sensitive personal data regarding the respondents were collected. To protect the anonymity of the respondents, all prose responses to the free-text questions (questions 13, 15, 18, and 20) have been omitted from the shared dataset. Written informed consent was not sought due to the low-risk nature of the research.\n\nThe survey responses include authors from 102 journals in the Earth & Environmental Sciences portfolio, and the geographical distribution of responses was similar to that of authors in the portfolio. Therefore, we can be reasonably confident that our responses are representative of Taylor & Francis authors in our Earth & Environmental Sciences journals.\n\nConfidence intervals have been calculated for certain parts of our analysis where we are comparing groups of different sizes within the survey, and we are only reporting on differences that are statistically significant. Microsoft Excel was used to prepare the tables and charts. Confidence intervals were calculator by using the Creative Research Systems sample size calculator14.\n\nA note about error bars and statistical significance. The country-comparison charts presented in this report include error bars, which plot the confidence intervals for the percentages shown. When making comparisons, error bars are useful as a visual means of demonstrating the range that likely contains the true overall value for each country in the chart. If the error bars for two or more countries overlap, we should be cautious about making substantive conclusions about any differences, because they may not be statistically significant. Therefore, only clearly statistically significant differences are included in the comparisons presented herein.\n\n\nResults\n\nWhy do researchers undertake the research that they do? It is a fundamental question and the answer is multifaceted, varying by career stage, geography, and subject discipline. However, the publication of the SDGs by the UN in September 2015, which had the stated aim of providing a âa shared blueprint for peace and prosperity for people and the planet, now and into the futureâ3, allows us an opportunity to frame the question in such a way that gets to the core of what researchers hope to achieve through their work, that is: âdo researchers study topics that contribute, either directly or indirectly, to the tackling of real-world problems?â.\n\nThe contribution of research. Comprising such urgent needs as Clean Water and Sanitation (SDG 6) and Climate Change (SDG 13) and tackling threats to Life on Land (SDG 15) and Life below Water (SDG 14), one might readily anticipate that a high proportion of research in the Earth and Environmental Sciences would have a part to play in meeting the needs expressed by the SDGs. Indeed, 74% of respondents indicated that their research contributed (directly or indirectly) to the tackling of real-world problems, such as those expressed by the UN SDGs (Figure 1). Furthermore, overall, 90% of respondents indicated that their work either currently contributed to meeting real-world problems or that it would be a priority for them in the future. Therefore, we might infer that, at least in the Earth & Environmental Sciences, it is a strong research imperative for our authors that their work contributes to the tacking of real-world problems.\n\nProportion of respondents whose research contributes to tackling real-world problems, now (A) and in the future (B).\n\nSuch a high percentage aligns with the voices of our journal editors, who, in contributing to our recent publication âSustainable Development Goals in the Earth and Environmental Sciencesâ15, expounded the variety, breadth, and richness of research that their journals and subject areas have to offer in tackling the challenges laid out in the SDGs.\n\nIn our survey, whilst younger researchers were slightly more likely to undertake this type of research (76% of respondents aged under 50 answered âYesâ compared with 70% of respondents aged 50 or older, with resolved confidence intervals), the difference was not very pronounced, thus suggesting that this is a multi-generational aspiration, rather than one solely driven by early-career researchers.\n\nTo understand a bit more about the motivating factors that sit behind the decision of our researchers to investigate topics that have application to real-world problems, we asked âWhy have you chosen to undertake research that contributes to these topics?â (Figure 2). The responses to this question presented a clear split between internal driversâpersonal interest (62%) and the desire to contribute to addressing real-world problems (78%)âand external drivers, such as encouragement from a university, other collaborators, or improved opportunities to secure research funding, with internal drivers and aspirations being the greater motivators.\n\nWe find it surprising that the influence of funders (15%) and institutions (16%) was only narrowly more influential than coincidence (14%) in prompting research that is skewed towards meeting these global challenges.\n\nWe saw the greatest gap between aspiration and reality when we asked what type of impact was most important to respondentsâ with a maximum of three selections. The most-preferred type of impact was citations from within the same field (69%), over against contribution to the advancement of research (53%), contribution to tackling âreal-worldâ problems, such as those expressed by the UN SDGs (21%), and input into policy decision-making (19%; Figure 3).\n\nInterestingly, having seen the strong desire of authors to undertake research with real-world application in the earlier questions, when compared with other types of impact, contributing to the tacking of real-world problems dropped to fifth in the list (21%), behind citations from within the same field (69%) and from adjacent/other fields (25%), and achieving a large readership (34%).\n\nWe note that some respondents may have felt that citations were a necessary step in contributing to the advancement of research or in tackling real-world problems, through knowledge sharing and discussion, as the reasons for these selections werenât probed further. However, as the question asked what the most important type of impact was to the researcher (âto youâ), we think this is unlikely to be a significant line of thought.\n\nInput into policy decision-making, âwhere the rubber meets the roadâ for much of the national-scale change that is required to meet the needs captured by the UN SDGs (19%), placed further down the list, on par with forming new collaborations (19%). Only attention from the press (3%) and attention on social media (3%) ranked lower.\n\nFeedback from respondents has indicated two key points: the aspiration of researchers to contribute to the tackling of real-world problems with their work, compared with a focus on citations as the key measure of impact. What role might the choice of a journal have to play in serving either of these aims? To investigate this further, we asked all of the respondents why they submitted their paper to the journal their work was published in, and also whether this journal was their first choice (Figure 4).\n\nThe predominant factor in determining the selection of a journal was its relevance to the authorâs research, and 64% of respondents indicated this was one of the most-influential factors underpinning their choice of journal. Interesting, reaching a broader non-academic audience (which we might link to the desire to contribute to resolving real-world challenges) came quite far down the list, with only 6% of respondents noting that this was an influential factor in their choice of journal.\n\nOn asking our authors whether the journal that they published in was their first choice, 75% indicated that it was, whilst 19% indicated that it was their second choice and 6% indicated that they had submitted their paper to more than one other journal before publishing it.\n\nIn this context, it is perhaps unsurprising to see that a journal having an Impact Factor was important to 42% of respondents. Interestingly, although 42% of authors indicated that the journal having an Impact Factor was an important factor in their decision-making, only 8% indicated that they chose the journal because it had the highest available Impact Factor, thus indicating that the presence of an Impact Factor was more important than the score itself. Many institutions, policymakers, and funders are keen to reduce emphasis on the Impact Factor as part of research assessment practices16, so there is perhaps a misalignment in the priorities of researchers, as opposed to their institutions and funders.\n\nTo investigate if (and if so, how effectively) publishers support the essential profile-raising and network-building needs of our research communities, we asked survey respondents about the opportunities for network-building and recognition that publishing in the journal had afforded them since they published their work.\n\nWhen asked if the publication of their article led to the formation of new connections with a range of different groups, almost half of respondents (46%) indicated that the publication of their article led to their forming new connections with researchers/research groups in their own country, whilst 35% formed new connections with researchers/research groups from other countries (Figure 5). From this feedback, we infer that publications and journals have a vital and valuable role in facilitating global knowledge sharing within subject communities.\n\nSurvey feedback highlighted the positive influence that journals and publishers can have in forming new academic collaborations. It is important to note, however, that this âmatchmakingâ effect does not appear to be as profound in non-academic contexts, with only 7% of respondents noting that they were approached about non-academic collaborations after the publication of their work (however, 48% of these respondents did note that their work influenced or greatly influenced this approach). Furthermore, only 10% of respondents formed new connections with practitioners, consultancies, or other non-academic communities as a result of publishing their work; only 8% formed new connections with governmental/inter-governmental policy-making bodies; and fewer still formed new connections with industry (7%) or funding bodies (5%).\n\nAs shown in Figure 6, one third of respondents (33%) indicated that, since the publication of their article, they had been approached regarding a potential research collaboration, with two thirds of these respondents noting that publication of their work influenced or greatly influenced this approach (Figure 7). In this regard, publishers and journals play a vital matchmaking role in linking together researchers for further collaboration.\n\nBased on survey feedback, authors based in different regions appear to place different emphases on the criteria that shape their choice of journal, and their views around impact.\n\nUnited States. Fewer respondents based in the United States indicated that having an Impact Factor was an important criterion in determining their choice of journal compared to the overall average (23% vs 42%). This same lower emphasis on the Impact Factor is seen in our post-publication author survey, which is sent to authors in all subject areas and all geographies, with respondents from the US rating this as a less-important factor in determining their choice of journal compared with the global average (Figure 8)17.\n\nInstead, US-based authors placed more value on real-world types of impact than the global average, with a higher proportion indicating that contribution to tackling big real-world problems, such as those expressed by the UN SDGs, was one of the most-important types of impact to them (29%), and a much-higher proportion indicating that having an input into policy decision-making was important to them (34% vs 19% overall; Figure 9).\n\nRecommendation from a colleague (39% vs 26% overall) and the journalâs capacity to reach a broader non-academic audience (13% vs 7% overall) were also deemed to be much more important factors as a means of identifying a suitable journal for US-based respondents, compared to the global average (Figure 10).\n\nChina. Responses from researchers based in China largely reflected the overall results, both in the most-important types of impact to them and the most-important factors that influence their choice of journal. As in other territories, respondents from China indicated that receiving citations from within the same field was one of the most-important types of impact for them (72%), followed by contribution to the advancement of research (49%) and readership/downloads (33%).\n\nHowever, one noticeable distinction was the relative unimportance of having a real-world impact in terms of contribution to tackling real-world problems (10% vs 21% overall; Figure 11) and input into policy decision making (8% vs 19% overall), perhaps because China-based respondents were less likely to have collaborations with groups who were involved in SDG-related activities (8% vs 16% overall; Figure 12).\n\nConversely, for China-based researchers, the relevance of a journal to their work was a much-more-important consideration (83%) compared to the global average (65%) and compared to authors based in the US (59%) and Europe (49%), whilst whether the journal had an Impact Factor was as important to Chinese respondents (44%) as the overall average (42%; Figure 13).\n\nUK and Europe. Respondents from the UK and Europe closely followed the global averages for both the types of impact that were most important and the most-important factors in determining the choice of journal.\n\nRespondents from the UK and Europe indicated that receiving citations from within the same field was the most-important type of impact to them (73%), followed by contribution to the advancement of research (50%) and readership/downloads (33%). Where respondents based in the UK and Europe differed was in the prospect of forming new collaborations (24%), which they considered to be a more-important type of impact than for respondents from India (16%) and China (14%; Figure 14).\n\nInterestingly, and perhaps related to the premium placed on network-building outside of their own subject communities, only 49% of respondents from the UK and Europe said that their work was published in the most-relevant journal, much lower than all other territories (65% average; Figure 15).\n\nIndia. Respondents from India again closely followed the global averages in terms of the types of impact that were most important and the most-important factors in determining the choice of journal. However, there were two distinct points of divergence.\n\nCompared to the global average, respondents from India placed significantly greater importance on the relevance of the journal for their work (87% vs 65% overall), comparable to respondents from China (83%) and significantly higher than respondents from the US (59%) and the UK and Europe (49%). Similarly, respondents from India placed much greater importance on the journalâs capability to reach their community (30%) compared to respondents from China (12%), the US (15%), and UK and Europe (18%), as well as to the overall average (19%; Figure 16).\n\nPerhaps most importantly, respondents from India indicated that a journalâs capacity to raise their profile was much more important to them (31%) than respondents from the other territories that we considered (UK and Europe 16%, China 14%, US 12%; Figure 17).\n\nAcademic publishing is multifaceted, with a range of different stakeholders located all around the globe, across both the private and public sectors. We asked participants the following question: How could journals or publishers help research to influence the response to real-world problems? Answers were provided as free text and clustered around four main improvements to mechanisms around: access, accessibility, communication of outcomes, and timeliness.\n\n1. Improve access to the latest research, in particular to non-academic/policy-maker audiences, as well as to the underlying code/data.\n\nâEngage closer with non-governmental organisations (environmental and social) â provide greater access to these organisations that are fundamental to achieving the SDGs but do not have the financial resources to enjoy access/membership of the Journals.â\n\nâProvide access to interesting real-world data setsâ / âPublish code and data along with papers; special issues focused on practical applicationsâ\n\nIn order to engage a non-academic audience, our respondentsâ views are clear: policy-makers, industry, and the wider public must have access to the original research, both the underlying data and the conclusions. In this regard, greater support for open access publication models18 across all key stakeholders is an important step to take to allow non-academic readers to engage with the latest research.\n\n2. Improve accessibility of research by changing the language, style, and format of publications to serve a non-academic audience.\n\nâPrepare readersâ digest versions of relevant articles, in multiple languages.â\n\nâProvide a policy-type document for research papers that tackle real-world problems. Original research paper may be difficult to read by policy-makers.â\n\nâPublish an e-digest of abstracts indexed by problem area. Send it to NGOs and managers in government agencies so they can quickly find articles that are relevant for their issues.â\n\nâIncreased use of executive summaries from research papers that are accessible to a broader audience than academiaâ\n\nâprovide support producing infographics and sharing research to non-academic audiencesâ\n\nTo help realise the potential reach, impact, and policy application of research, respondents noted that research outcomes should be presented in a format, style, and language that is accessible and comprehensible to a non-academic audience. Whilst the research article well-serves the research community, the structure, tone, and length may create some barriers for non-academic readers, who are often looking for evidence pertaining to their particular point of need and may be put-off from drawing out points of relevance from a full research paper.\n\n3. Improve communication links to raise the visibility of research implications on policy and real-world issues.\n\nâMaking more publicity to the \"non-scientific world\" of the issues that are published in the journalsâ / âbe present at policy eventsâ\n\nâSpecial editions and workshops (can be via Zoom) to bring people together.â\n\nâShare published papers on social media and create TV shows where scientists engage on current issues.â\n\nâConnections with academic media outlets, like the Conversation etc.â\n\nâThey should announce research grants related to real world problemsâ\n\nAuthors and publishers need to maximize the opportunity to bring the latest research into the public conscious, with the aim of cultivating a culture that drives policy change. Respondents noted that non-academic summaries, workshops, and discussion forums could directly engage with policy-makers right at the point of need. However, as noted by one respondent, it is also important for publishers to âbe presentâ where appropriate at policy events and to advocate for the value of the research that they publish on behalf of their authors.\n\n4. Better support the publication of research on areas of particular relevance to live policy issues.\n\nâSeek out authors who are also practitioners.â / âBy opening spaces for discussion among different actors (policy-makers, civil society and academia) and societal sector.â\n\nâBe willing to publish applied work, not just academic studies.â / âencourage and publish more transdisciplinary researchâ\n\nâBy staying focused on their journals' scope which should be specific to these real-world problemsâ\n\nâBy planning special issues which focus on research that are in response to real-world problems. When doing so, ensuring that enough time is given for research in this area to be specifically conducted, and not expecting that data is already available to be tailored into a paper that addresses these issues.â\n\nâBy considering articles that address real world problems, even it if they are not considered \"high impact\" or \"potentially citable\".â\n\nThese comments collectively strike right at the heart of the purpose of research journals.\n\n\nDiscussion\n\nOverall, 90% of respondents indicated that their work either currently contributed (directly or indirectly) to meeting real-world problems or that it would be a focus for them in the future. However, when asked about forms of impact, citations were viewed as more important than advancing research or contributing to the SDGs.\n\nWe might infer that this focus on citations as the key form of impact is driven by the mechanisms that underpin research assessment. There are calls to move away from a focus on citations and publication venue, and to judge work based on its own merits19. However, changes to institutional assessment mechanismsâand academic culture itselfâare slow to take effect. This focus on citations may be compounded by the ways in which impact is judged at a national level, with judgements around research âexcellenceâ by country often based on comparing field-weighted citations from one country to another20.\n\nIs this an issue worth addressing? The answer to this question must be âyesâ, both for the research and then for the researcher, institution, funder, and publisher. If the measures that exist within academic publishing remain unchanged, continuing to prioritize other metrics and outcomes, we risk devaluing the necessary application of original research to addressing our global challenges. It is then a slippery slope from devaluing to deprioritising to not doing at all, and the devaluing of important, consequential research today will likely lead to less of it in the years to come, at a time when much more research is required to help meet our global societyâs needs, not less.\n\nThere is also a cultural issue. If research as a whole does not pursue greater public engagement and support the tackling of our global challenges, the research community risks appearing elitist and out-of-touch with the public conscious21.\n\nHow might we bridge the gap? There is clearly a high level of engagement by the respondents in contributing to the thinking around real-world problems. However, at the heart of academic research, there are seemingly competing interests, which push back on our authorsâ desire to tackle the key challenges affecting our society, and instead pull authors to pursue volume of output and accruing citations. Therefore, given this apparent disconnect between the ambitions of researchers to address real-world problems with their work and the realities that drive their choice of journal and preferred type of impact, we next turn to what might be done to bridge this gap.\n\nThere seems to be a knowledge gap for our researchers in understanding and communicating the link between their individual, highly focused projects and wider live policy issues, such as those expressed by the SDGs. Respondents encouraged publishers to facilitate the connection of an individual output to a real-world challenge, such as the SDGs, through the publication of summary research conclusions in approachable language (âlay summariesâ, âpolicy highlightsâ, or similar), either alongside or as part of the research article, and to work with researchers and institutions to explore new and alternative ways of disseminating such summaries to the general public or a policy audience.\n\nAuthors should be encouraged to articulate how their work contributes to real-world challenges, such as how the work aligns with a particular SDG (goal or target). An example of this is the European Commissionâs Horizon Results Platform, which allows authors to identify their work with particular SDGs and to flag research outcomes as âClaiming significant policy influenceâ22. Publishers and journal editors should be proactive in this process through the curation of special issues that are directly linked to live policy-relevant issues, such as those expressed in the Horizon Europe missions, to encourage greater consideration of policy priorities by our researchers. In addition, a focus on interdisciplinarity and the policy relevance of work should be encouraged, as our respondents have indicated that these are essential to ensuring that research has a real-world impact.\n\nTo support this work, we will work with our editors and society partners to introduce policy-impact statements widely across our Earth & Environmental Sciences journals over the next 12 months. We hope that this approach will facilitate the contextualisation of new research within larger global needs and aid in the impact of that work on policy decision-making. We will additionally hold a cross-stakeholder discussion forum in 2021 to explore other appropriate structural ways of clarifying policy relevance (such as to particular SDGs) at an article level. We will also publish cross-portfolio special issues on policy-relevant topics, beginning with the policy priorities expressed in the European Commissionâs Horizon Europe missions23, to be published on World Earth Day 2022. By doing this, we aim to encourage the greater consideration of policy priorities by our researchers and to support the publication of such research within our journals. Finally, we will continue to transition our Earth & Environmental Sciences journals onto more-open data-sharing policies to support the availability, reuse, and citation of codes and data24.\n\nInstitutions could consider developing training programs for their researchers to help them think through how a specialised piece of work can have wider implications, such that it can be incorporated into policy change, and how researchers can communicate and demonstrate the relevance of their work to those discussions.\n\nComments from respondents suggest that changes to traditional mechanisms around engagement, knowledge transfer, and research assessment are required to create better links and lines-of-sight between research endeavour and policy development. Furthermore, several respondents also felt that addressing real-world problems with their work was not enough in itself to contribute to change; rather, action by policy-makers was a decisive factor in whether their research would have such an influence.\n\nTo enthuse researchers with the ambition of addressing real-world impact, they need to become more cognisant of how their research is incorporated into policy advice and decision-making. When asked whether, since the publication of their work and to the best of their knowledge, their article had been used or referenced in a non-academic output, the majority of respondents (32%) answered âdonât knowâ, and only 10% suggested that their work had been used in a policy document. At present, authors canât easily track and often arenât aware if/how their work is used outside of other research articles. Therefore, there needs to be a much-more-robust feedback mechanism from governments, NGOs, lobbyists, and advisory groups to the academic community, e.g. through the expansion of tools such as Altmetric25, if researchers are going to feel sufficiently equipped to engage in policy advocacy and to feel that non-academic outputs, such as those suggested above, would be valued and acted upon.\n\nUniversities are well-positioned to educate their faculties about the mechanisms and approaches open to them for communicating their work to a policy audience. It is in the interest of institutions and funders to encourage researchers to engage in policy and advocacy. Research offices can leverage the resources already out there to help with guidance and training, and could consider partnering with groups such as Sense about Science and the UKâs Parliamentary Office on Science & Technology (POST) to share insights from those with close connections to legislators. In collaboration with these groups, Taylor & Francis offers guidance to help published authors on getting their research into the UK Parliament26, as well as supporting the work of Vitae on impact and evaluation27, and we are continually expanding these resources.\n\nMuch has been done to support the translation of research conclusions into language suitable for a non-academic audience, through platforms such as Kudos28, or through social media and academic news services such as EurekAlert!29 and the Conversation30. However, such activity has yet to become common practice. The translation of research results into âpolicymaking languageâ to make it useable for policy-makers might be achieved through the publication of accompanying abstracts for a non-academic audience or policy implications/highlights for each new piece of work. Ensuring that there are channels for research to reach policymakers is another critical activity publishers should consider. Publishers might consider synthesising and facilitating meta-comparisons of similar research outcomes to support a streamlined evidence-based policymaking process. New products or services could be created to assist in this translation and dissemination activity, as well as the presentation of relevant research to policy-makers and their advisory groups, such as TrendMD31. These services will however require financial support; additionally, researchers need to be incentivised to ensure that they are positively rewarded for making these connections. Such activities must be supported by training and incentives from those host institutions keen to ensure that their research outputs have a real-world impact. To support this activity, publishers and funders should facilitate interoperable standards and persistent identifiers to ensure that all of the outcomes are linked, and that policy decisions are informed by a rich and networked research base.\n\nMost broadly, there may be a need to extend the âresearch cycleâ to incorporate a policy dimension into the initial stage of developing the research question, thereby ensuring that researchers review funding calls in their area and study the research agenda of their governments and policymakers for activities relevant to their field. The European Geosciences Union (EGU) have published a helpful graphic, which outlines the interplay between policy and the research cycle32. Greater adoption of this âpolicy cycleâ approach, including the formalisation of prompts at the dissemination stage of the research cycle to direct relevant outcomes to policy-makers, could help to influence sharing behaviours, and the expansion of a researcherâs existing network into the policy arena.\n\nPublishers might typically express the value that they provide to the academic community through the oversight of rigorous peer-review processes to validate research conclusions and through the curation, dissemination, and preservation in perpetuity of the academic record. However, based on the responses to the questions âDid the publication of your article lead to you forming new connections with any of the following?â and âHave any of the following happened in your career since the publication of your article?â, it is clear that publishers also play a hugely important role in facilitating new collaborations between researchers, which are often triggered by research publications, with collaboration a critical component of research success33.\n\nIn addition, through the promotion and dissemination of their research, publishers have the capability to foster meaningful engagement with the general public through press/news media coverage or mentions on social media. Publishers serve an important role as bridge-builder between communities both within and without academia, and should ensure that they continue in this function.\n\nRespondents raised concerns around working in small silos, with limited personal networks and small spheres of influence, particularly in the policy space, which held back the impact of their research in tacking real-world problems. Our survey highlights some areas where publishers should consider how to better connect researchers with the wider non-academic community to help their work resonate outside academic circles, and to have real-world impact, including network-extending activities with non-academic communities, specifically policy-makers and industry.\n\nWe suggest that universities give thought to helping their researchers to better grasp the mechanisms for how their research can influence policy, and what steps they can personally take to advocate for the uptake of their conclusions into the policy debate. Likewise, we encourage funders, universities, and publishers to work with governments and other pertinent stakeholders to develop a more robust feedback mechanism to the academic community so that researchers can understand how their work is used in decision-making and can feel equipped to advocate for a real-world impact with their work.\n\nOur feedback suggests that research assessment practices should be reviewed and revised to ensure that policymakers, institutions, and funders capitalise on the aspirations of researchers to effect real-world change and reward those aspirations. Revised practices should be supported by incentives that place greater emphasis on clarifying policy impact in grant applications, and deliver greater recognition for policy impact. In this regard, the Final Report of the Expert Group on Indicators for Researchersâ engagement with open science34, the Open Science Policy Platformâs final report35, and LERUâs Open Science and its role in universities: a roadmap for cultural change report roadmap for institutions36 all include excellent recommendations, and also show how moves to change research assessment practices dovetail with calls to increase open science / research practices.\n\nOur authors expressed a desire to conduct research that helps to tackle global needs, and most are already actively achieving this with their research. However current assessment frameworks continue to focus on and reward citations and publication in highly ranked journals at the expense of opportunities for impact or other forms of output. As part of our ongoing training and support for our authors and editors, we highlight the role of research metrics in measuring performance, but also their limitations, and note that such tools need to be used appropriately, as part of a âbasket of metricsâ, and not in place of a qualitative review of individual outputs37,38. We are also investing in diversifying research outputs, including providing support for non-traditional formats, such data notes and software tool articles39. We have committed to making a broad set of metrics available across journals that provide a richer overview of their performance, linked with guidance to contextualise these data.\n\nWe suggest that institutions reconsider the performance-assessment frameworks that they use, instead placing greater value on a broader range of research outputs, such as patents, case studies, and engagement with secondary education. Likewise, we suggest that funders continue to facilitate, through the grant applications that they support, the pursuit of research with clearly defined opportunities for policy-relevant outcomes or other tangible real-world impact, such as the Gates Foundationâs support of the Grand Challenges for Global Health (GCGH) initiative40,41.\n\n\nConclusion\n\nFollowing a survey of >2,500 researchers who had published in our Earth & Environmental Sciences journals portfolio, we found that a majority of respondents (90%) indicated that their work either currently contributed to meeting real-world problems or that it would become a priority in the future, thus suggesting that, as one might anticipate, the tackling of real-world challenges is a significant research priority in the Earth & Environmental Sciences.\n\nWhilst it is very encouraging to see that the majority of research in the subject area is concerned (directly or indirectly) with addressing our global needs, the impetus seems to be altruistic researcher desires, rather than incentives or support from publishers, funders, or institutions. As a result, it seems that this laudable ambition is being lost amidst the realities of being a researcher â where success is predominantly measured by citations and publication venue.\n\nTherefore, herein, we have used survey responses to consider what opportunities we have as a research community to collectively assist researchers in having the real-world impact with their work that they (and we) would like it to have. Accompanying this report is a set of suggestions for the wider community, which have been drawn out of the conclusions from this work, along with a series of commitments which we as Taylor & Francis will take to play our part in addressing some of these issues.\n\nWe welcome feedback from the community and opportunities for collaboration, and we anticipate that these recommendations will be further refined as we implement our commitments and undergo further consultation.\n\n\nData availability\n\nFigshare: Taylor-and-Francis_Impact-Assessment-of-Earth-and-Environmental-Sciences-Research-Author-Survey_Raw-Data_Figshare, https://doi.org/10.6084/m9.figshare.13281146.v142.\n\nFigshare: Taylor-and-Francis_Earth-and-Environment-Survey-Questions, https://doi.org/10.6084/m9.figshare.13281104.v113.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Author information\n\nDr Andrew Kelly holds a BSc (First Class Honours) in Chemistry from the University of York (2003) and a PhD in Chemistry from the University of Bath (2008). He has worked in publishing for more than 10 years and is currently a Journals Portfolio Manager at Taylor & Francis, where he is responsible for the Remote Sensing, GIS & Cartography titles. ORCiD: 0000-0002-5785-4782\n\nVictoria Gardner is Director of Policy at Taylor & Francis, where she advises on global policy developments around scholarly communications including around open access, copyright and IP, and the move to digital. She has worked in a variety of roles within Taylor & Francis, including portfolio management, open access, and technology. ORCID: 0000-0002-8519-6377\n\nAnna Gilbert holds a BTh in Theology from Bangor University and an MSc in Social Research Methods from the University of Surrey. She is currently a Research & Analytics Manager at Taylor & Francis, where she is responsible for primary research and marketing/web analytics. ORCiD: 0000-0002-9627-9274\n\n\nAcknowledgements\n\nThe authors thank Tom Fleet for his support in the set-up and execution of the survey.\n\n\nReferences\n\nfor example, see the Lisbon Strategy and http://uis.unesco.org/apps/visualisations/research-and-development-spending/.\n\nhttps://web.ornl.gov/sci/techresources/Human_Genome/project/economics.shtml.\n\nUnited Nations Sustainable Development Goals. 2015. Reference Source\n\nhttps://ec.europa.eu/info/horizon-europe-next-research-and-innovation-framework-programme/#missions-in-horizon-europe.\n\nThe Lund Declaration. 2009. Reference Source\n\nhttps://www.london.edu/think/who-needs-experts, accessed 30 October 2020.\n\nWellcome Global Monitor. 2019. accessed 30 October 2020. Reference Source\n\nhttps://blog.okfn.org/2020/05/05/brits-demand-openness-from-government-in-tackling-coronavirus/.\n\nhttps://www.wissenschaft-im-dialog.de/projekte/wissenschaftsbarometer/wissenschaftsbarometer-corona-spezial/.\n\nhttps://www.ref.ac.uk/.\n\nhttps://www.theimpactinitiative.net/.\n\nhttps://editorresources.taylorandfrancis.com/peersupport/research-impact-author-survey/.\n\nResearch & Analytics, Taylor & Francis: Taylor-and-Francis_Earth-and-Environment-Survey-Questions. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.13281104.v1\n\nhttps://www.surveysystem.com/sscalc.htm. (accessed 07 October 2020).\n\nhttps://doi.org/10.6084/m9.figshare.12933173.\n\nSuggestions for a National Framework for Publication of and Access to Literature in Science and Technology in India. Appendix 3, point 1. Accessed 27 October 2020. Reference Source\n\nTaylor & Francis Journals Author Survey (results not publicly available).\n\nhttps://www.tandfonline.com/openaccess.\n\nhttps://sfdora.org/.\n\nOverall output of select geographical group comparators and related FP7- and H2020- funded publication output. European Commission. 2017. Reference Source\n\nhttps://wellcome.org/sites/default/files/public-first-advocating-rd-investment.pdf.\n\nhttps://ec.europa.eu/info/funding-tenders/opportunities/portal/screen/opportunities/horizon-results-platform.\n\nhttps://ec.europa.eu/info/horizon-europe/missions-horizon-europe/mission-boards_en.\n\nhttps://authorservices.taylorandfrancis.com/data-sharing-policies/open-and-fair/.\n\nhttps://www.altmetric.com/.\n\nhttps://authorservices.taylorandfrancis.com/getting-your-research-into-parliament/.\n\nhttps://www.vitae.ac.uk/.\n\nhttps://info.growkudos.com/.\n\nhttps://www.eurekalert.org/.\n\nhttps://theconversation.com/uk.\n\nhttps://www.trendmd.com/.\n\nhttps://www.egu.eu/policy/basics/cycle/.\n\nInternational research collaboration, an activity that is generally associated with greater citation impact: p4, INTERNATIONAL COMPARATIVE PERFORMANCE OF THE UK RESEARCH BASE 2016. Reference Source\n\nhttps://ec.europa.eu/research/openscience/index.cfm?pg=altmetrics_eg.\n\nhttps://ec.europa.eu/research/openscience/index.cfm?pg=open-science-policy-platform.\n\nhttps://www.leru.org/publications/open-science-and-its-role-in-universities-a-roadmap-for-cultural-change.\n\nhttps://authorservices.taylorandfrancis.com/research-impact/.\n\nhttps://editorresources.taylorandfrancis.com/understanding-research-metrics/.\n\nhttps://f1000research.com/articles/9-657.\n\nMatthews KR, Ho V: The grand impact of the Gates Foundation. Sixty billion dollars and one famous person can affect the spending and research focus of public agencies. EMBO Rep. 2008; 9(5): 409â412. PubMed Abstract | Publisher Full Text | Free Full Text\n\nhttps://www.gatesfoundation.org/How-We-Work/Quick-Links/Grants-Database/.\n\nResearch & Analytics, Taylor & Francis: Taylor-and-Francis_Impact-Assessment-of-Earth-and-Environmental-Sciences-Research-Author-Survey_Raw-Data_Figshare. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13281146.v1"
}
|
[
{
"id": "77703",
"date": "10 Feb 2021",
"name": "Brooks Hanson",
"expertise": [
"Reviewer Expertise Earth and space science broadly",
"and scholarly publishing"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview of Andrew et al., The disconnect between researcher ambitions and reality in achieving impact in the Earth & Environmental Sciences â narrowing the gap.\nThis is a report of an interesting survey getting at a major question related to understanding motivations of researchers in conducting and publishing research, and ultimately how to align incentives to support, recognize, and reward better research and activities of scholars aimed at addressing societal challenges and working with communities.\nMajor points:\n1) The main improvement needed for this paper is placing it in context of other work and author surveys. There are many related and similar surveys and analyses, done by publishers, societies, funders, and scholars, and none (not exaggerating; none) are cited or mentioned. Much of the findings here regarding citations and priorities around publishing, open access, and more, have been covered in other recent author surveys, in this general discipline and other disciplines. This context is essential for this paper to be considered scholarly (and published in a scholarly journal). Iâve reviewed a lot of papers over the years, and this is the first submitted to a leading journal where Iâve seen such a lack of referencing. This might be acceptable for a report by a publisher (cf. Elsevierâs recent gender analysis, self-published, also completely without references) but not a submission to a scholarly journal. Most of the references are just to websites, not any formal survey results or scholarly research on these topics (thereâs a lot even in the past few years). Such comparisons would also strengthen some of the conclusions.\nJust a note that JpGU and AGU have conducted a somewhat similar survey of their members. The results are not published yet but were presented in this session: https://agu.confex.com/agu/fm20/meetingapp.cgi/Session/105702 at the recent AGU Fall Meeting (see presentation starting at about 40 minutes; registration is required). Iâve been involved in helping this survey. Overall these results (and others AGU has conducted but not published) are similar to the results given in the later questions here regarding selecting journals, citations, etc. However, on the motivation for research (first question in this survey, and the one that sets that main stage for discussion), the AGU-JpGU wording was different but a large number of respondents, well beyond a majority, indicated that their primary motivation for research was around basic âdiscoveryâ or âelaboration/synthesisâ rather than âresponding to responsibility of society.â JpGU members even moreso. In this survey, unlike the T&F one, there was a large age-related difference between early career, and later-career respondents (early career researchers were more focused on topics related to societal impact). I suspect that the populations of respondents overlap heavily in the two surveys. Recognizing that the AGU-JpGU results are not yet fully analyzed or published, Iâm just raising this to bring caution to over interpreting the first question of this survey as worded. This question is, however, the most interesting one to explore and provides much of the interesting novelty here. Just be cautious in interpreting the answers.\nOne test would be also to simply score recent publications (outputs) as to whether they align with the resultsâthat is, do most of the outputs directly or indirectly support SDGs, for example? My sense is that in the Earth and space sciences, many indirectly do, but that the path is long and Iâm not sure 75% would without quite a stretch.\n2) As the authors note there is a disconnect between authors reporting that they are working (directly or indirectly) on societal relevant topics vs. the âimpactâ (that is, citations) that they are seeking in their work. Further exploration is needed on whether the respondents misinterpreted the first question or if it was worded so vaguely (âindirectlyâ) as to be meaningless. Note that much âbasicâ research in the Earth, environmental, and space science has widespread indirect impacts. Much real time âbasic-scienceâ data about the Earth is used in the GPS system, weather predictions, or other uses, e.g. For examples, see this discussion here that I was involved with: https://eos.org/editors-vox/earth-and-space-science-for-the-benefit-of-humanity and the linked papers. Indeed many grant applications require a statement regarding impacts.\nSimilarly, results for the question on impact expected by the authors are used in comparison. I also wonder if the wording and reality of the scope of published papers drove this response (that is, many have an indirect vs. direct impact) and the response was viewed as a direct impact.\n3) The authors list a number of actions T&F are taking or should take. Interestingly, T&F has not signed DORAâas Springer-Nature and Elsevier have now signed (whatever one thinks of that), Wiley and T&F are the major publishers who have not (many individual society journals published with Wiley have). Perhaps the authors could indicate why or why not that would be appropriate and how to leverage that impact. Hereâs a recent editorial from a T&F publication: https://www.tandfonline.com/doi/full/10.1080/10919392.2018.1522774\n4) The authors indicate what some stakeholders, especially publishers, might do. In the Earth environment and space sciences, there are several leading global societies. These are not mentioned. What is their role? Many have missions aligned with providing benefits to society and many are involved in science communication, policy, outreach and training/mentoring (moreso than most commercial publishers and indeed universities). Indeed this might be an argument to focus on publishing with a society versus a commercial title, where these resources are more directly leveraged.\nOther items:\nThe authors argue that it is surprising that JIF is important to researchers but that they donât always/regularly choose the highest JIF journals when submitting. This is because researchers know rejection rates and do optimization around likelihood of success (or they donât want to waste their time, which is also important).\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "8459",
"date": "08 Jul 2022",
"name": "Andrew Kelly",
"role": "Author Response",
"response": "Responses to the reviewer comments have been added in italics. Reviewer's comments: This is a report of an interesting survey getting at a major question related to understanding motivations of researchers in conducting and publishing research, and ultimately how to align incentives to support, recognize, and reward better research and activities of scholars aimed at addressing societal challenges and working with communities. Major points: 1) The main improvement needed for this paper is placing it in context of other work and author surveys. There are many related and similar surveys and analyses, done by publishers, societies, funders, and scholars, and none (not exaggerating; none) are cited or mentioned. Much of the findings here regarding citations and priorities around publishing, open access, and more, have been covered in other recent author surveys, in this general discipline and other disciplines. This context is essential for this paper to be considered scholarly (and published in a scholarly journal). Iâve reviewed a lot of papers over the years, and this is the first submitted to a leading journal where Iâve seen such a lack of referencing. This might be acceptable for a report by a publisher (cf. Elsevierâs recent gender analysis, self-published, also completely without references) but not a submission to a scholarly journal. Most of the references are just to websites, not any formal survey results or scholarly research on these topics (thereâs a lot even in the past few years). Such comparisons would also strengthen some of the conclusions. Just a note that JpGU and AGU have conducted a somewhat similar survey of their members. The results are not published yet but were presented in this session: https://agu.confex.com/agu/fm20/meetingapp.cgi/Session/105702 at the recent AGU Fall Meeting (see presentation starting at about 40 minutes; registration is required). Iâve been involved in helping this survey. Thank you and we acknowledge the limitations of our introduction. As part of the refocusing of the article, we have pared-back the introduction to the article and included additional referencing to support the discussion. Overall these results (and others AGU has conducted but not published) are similar to the results given in the later questions here regarding selecting journals, citations, etc. However, on the motivation for research (first question in this survey, and the one that sets that main stage for discussion), the AGU-JpGU wording was different but a large number of respondents, well beyond a majority, indicated that their primary motivation for research was around basic âdiscoveryâ or âelaboration/synthesisâ rather than âresponding to responsibility of society.â JpGU members even moreso. In this survey, unlike the T&F one, there was a large age related difference between early career, and later-career respondents (early career researchers were more focused on topics related to societal impact). I suspect that the populations of respondents overlap heavily in the two surveys. Recognizing that the AGU-JpGU results are not yet fully analyzed or published, Iâm just raising this to bring caution to over interpreting the first question of this survey as worded. This question is, however, the most interesting one to explore and provides much of the interesting novelty here. Just be cautious in interpreting the answers. Thank you for raising this and we agree that it would be interesting to investigate further. One test would be also to simply score recent publications (outputs) as to whether they align with the resultsâthat is, do most of the outputs directly or indirectly support SDGs, for example? My sense is that in the Earth and space sciences, many indirectly do, but that the path is long and Iâm not sure 75% would without quite a stretch. This was a very interesting suggestion. We have used Dimensions SDGs category data (new Figure 2) to analyse their quantitative alignment of research published in the same set of journals with the SDGs and compared this with the authorâs qualitative responses. 2) As the authors note there is a disconnect between authors reporting that they are working (directly or indirectly) on societal relevant topics vs. the âimpactâ (that is, citations) that they are seeking in their work. Further exploration is needed on whether the respondents misinterpreted the first question or if it was worded so vaguely (âindirectlyâ) as to be meaningless. Note that much âbasicâ research in the Earth, environmental, and space science has widespread indirect impacts. Much real time âbasic-scienceâ data about the Earth is used in the GPS system, weather predictions, or other uses, e.g. For examples, see this discussion here that I was involved with: https://eos.org/editors-vox/earth-and-space-science-for-the-benefit-of-humanity and the linked papers. Indeed many grant applications require a statement regarding impacts. Similarly, results for the question on impact expected by the authors are used in comparison. I also wonder if the wording and reality of the scope of published papers drove this response (that is, many have an indirect vs. direct impact) and the response was viewed as a direct impact. A comment has been added on this in the revised submission to note further research would be useful to better understand the motivations and responses. 3) The authors list a number of actions T&F are taking or should take. Interestingly, T&F has not signed DORAâas Springer-Nature and Elsevier have now signed (whatever one thinks of that), Wiley and T&F are the major publishers who have not (many individual society journals published with Wiley have). Perhaps the authors could indicate why or why not that would be appropriate and how to leverage that impact. Hereâs a recent editorial from a T&F publication: https://www.tandfonline.com/doi/full/10.1080/10919392.2018.1522774 Pleasingly, Taylor & Francis has since signed DORA, but no comment has been made in the article, as the policy-related points have been removed. 4) The authors indicate what some stakeholders, especially publishers, might do. In the Earth environment and space sciences, there are several leading global societies. These are not mentioned. What is their role? Many have missions aligned with providing benefits to society and many are involved in science communication, policy, outreach and training/mentoring (moreso than most commercial publishers and indeed universities). Indeed this might be an argument to focus on publishing with a society versus a commercial title, where these resources are more directly leveraged. This was an oversight from the previous submission. As we have removed the policy discussion, we havenât elaborated on this further, but we acknowledge the mission focus of many of the leading societies and their importance in shaping the behaviours of researchers in their communities. Other items: The authors argue that it is surprising that JIF is important to researchers but that they donât always/regularly choose the highest JIF journals when submitting. This is because researchers know rejection rates and do optimization around likelihood of success (or they donât want to waste their time, which is also important). We agree that likelihood of success is one of the main drivers in the decision-making of authors when selecting a journal and included a paragraph on whether the article was finally published in the first/second/third-or-more choice. We also suggest that speed of publication/time to first decision, and the journalâs relevance to the community are other important drivers in addition to acceptance rate and Impact Factor."
}
]
},
{
"id": "79623",
"date": "12 Apr 2021",
"name": "Martin Dominik",
"expertise": [
"Reviewer Expertise Astronomy",
"science policy"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI am having some difficulties with the policy framing of the article, and it does not become obvious what point exactly the authors intend to make. A few statements donât appear to match up.\nThe article touches on many topics, but I feel that none of them are discussed to a sufficient extent. It initially centres around 3 key questions that are to be addressed by a survey of authors, but towards the end it morphs into an essay on developing an environment that supports translating research into policy, which is not much underpinned by the survey data. Consequently, the article feels like two that are loosely connected. Moreover, it sometimes reads like a policy statement and advertisement by Taylor & Francis rather than a research article. This impression is strengthened by the lack of research articles amongst the references in conjunction with the authors not adequately positioning their findings in the context of other research on the topic.\nThere is a fundamental conflict between the aspiration of âmaximising the capability of research to achieveâ and focusing efforts on addressing urgent needs, which remains unresolved in the article. Throughout, these are conflated and confused. I would consider âcapabilityâ a most relevant keyword in this context.\nSeveral references are ill-chosen for supporting the specific point that the authors try to make. Specifically, the Lisbon strategy (reference 1) has the declared aim âto make Europe the most competitive and dynamic knowledge-based economy in the world, capable of sustainable economic growth with more and better jobs and greater social cohesionâ, but it does not include an explicit call for research to have âimpactâ. Moreover, the report of reference 21 states that âPeople are broadly split on whether the UK invests too much in long-term R&D rather than solving issues that matter now (33% agree vs. 35% disagree)â. The link in reference 20 does not work.\nIt is rather unusual for me to defend Michael Gove, but âwe no longer need expertsâ is a (popular) misquotation by omission. He stated: âI think the people of this country have had enough of experts with organisations with acronyms saying that they know what is best and getting it consistently wrong.â, and the latter part of the statement is much relevant for scientists engaging in public debate. It is important to know how to build and maintain trust.\nWhile the authors elaborate on the term âimpactâ, it remains problematic and likely to be understood in various ways. One could challenge the statement about quantification in economic terms being straightforward, and in particular question whether benefits should be evaluated in such a way. For example, people dying early could be economically beneficial, but would that be societally desired?\nThe âmissionsâ approach in Horizon Europe is somewhat controversial. Notably, the recent 2020 Euroscience Open Forum (ESOF) included a session âDoes science for missions undermine the missions of science?â. Likewise, the authors state that the drive to solve \"the Grand Challenges of our time\" has acquired increased urgency during the COVID-19 pandemic, but one could also argue that prominently reveals a potential flaw of focusing on identified challenges, which is in neglecting those strands of research that are most suitable to provide the basis for the next challenges that we are to encounter (e.g. https://www.statnews.com/2020/02/10/fluctuating-funding-and-flagging-interest-hurt-coronavirus-research/ and https://www.nytimes.com/2021/04/08/health/coronavirus-mrna-kariko.html)\nThe authors refer to citations as the primary currency of success or progress, but it might be worth keeping in mind that it is a widespread myth that this applies universally. In particular, the quoted UK Research Excellence Framework (REF) is not based on citation counts. Moreover, there are significant differences between âimpactâ in the REF and âPathways to impactâ in the context of funding applications to UK Research Councils. Both are distinct from the âPathways to Impact initiativeâ (reference 11). I appreciate the authors mentioning a âchain reactionâ emerging from original research. Could one elaborate on what would determine the value of research?\nWith regard to the role of academic publishing, I note that the International Science Council has recently published an insightful report âOpening the record of science: making scholarly publishing work for science in the digital era.â1\nThe definition of the three main aims of the survey does not specify what group of people âour communitiesâ refers to. I am far less confident than the authors about the respondents being ârepresentativeâ. I would expect that those who just care about their bibliometric profile and other similar performance indicators are not inclined to spend any time responding, which would result in the respondents being more engaged for the scientific community and the wider society.\nI found the ânote about error bars and statistical significanceâ almost entirely stating trivialities, whereas the authors do not provide the crucial information of what the quoted âerror barsâ actually refer to and what they mean, which leave me unable to interpret them.\nThere are some substantial weaknesses with the survey questions and the provided answer options. It is somewhat confusing that in some cases respondents were able to pick any number of answers from a list, whereas in others the number of choices was limited. This poses some difficulties for the interpretation of the results and the limitations to answer options should be mentioned clearly in the respective figure and/or captions. It would also be useful if the authors referred to the question numbers.\nMy main concern with regard to the survey is about Q14 and Q16, which refer to âreal-word problemsâ. I think that it is an unfortunate choice that the authors put these central rather than referring to Q11 in conjunction with Q3 on addressing the question on why researchers undertake the research that they do. While the term âreal-world problemâ carries a polemic tone suggesting that academics might be detached from reality, âdirectly or indirectly contributingâ is remarkably fuzzy. It is not clear to me what Q14 and Q16 are actually able to capture, and I feel that answering with âyesâ, ânoâ, or âDonât knowâ is mostly a matter of interpretation of the question. I could make a case for my research falling into either of these categories, depending on what point of view I assume. In fact, âdonât knowâ appears to be a good option given that for some research the connection to âreal-word problemsâ is not immediately apparent and the connection might only be built in the future. Apparently, a substantial number of respondents chose that option. I also note that Q16 refers to âpriorityâ whereas Q14 does not. I did not see the authors commented on lower numbers for an affirmative response on Q16 as compared to Q14.\nI also wonder how many of the respondents are familiar with what the UN SDGs are, or are willing to look at up before they answer the question. I note that SDG 8 explicitly recognises creativity and innovation as drivers of economic growth, which aligns fundamental research, not directly targeted at specific challenges, with the UN SDGs.\nSomething that puzzles me is that 38% of the respondents did not choose the answer âI find researching these topics interestingâ. Why do they do research that they are not interested in?\nIt would seem to me that the survey reveals another âgapâ than the one the authors claim. A key gap appears to be in how the research actually materialises into something useful, with only about 20% of the survey respondents stated that âinput into policy decision-makingâ or âcontribution to tackling big real-world problems, such as those expressed by the UN SDGsâ was amongst the most important forms of âimpactâ (although they were limited to 3 answers). In contrast, the authors elaborate on the point that respondents ranked formal recognition over making a contribution and state that we risk devaluing the necessary application of original research to addressing our global challenges by prioritising other metrics and outcomes. However, their study does not provide evidence for that. If the research of the respondents is oriented towards âreal-world problemsâ, the underlying motivation is not the relevant issue. Unfortunately, the authors do not elaborate on to what extent âcontribution to the advancement of researchâ is aligned with âcontribution to tackling real-world problemsâ and/or âinput into policy decision-makingâ or rather not. It is the more unfortunate that respondents were restricted to a maximum of 3 answers for Q11 rather than being able to state where each of them ranks in priority.\nOn the question of why authors chose to submit their manuscript to the specific journal, the top chosen answer is pretty much an umbrella category that encompasses more than half of the other answer options, which are more specific on what most ârelevantâ means.\nThe authors should define what they consider âEuropeâ, e.g. if respondents stated that they are located in Turkey, have their answers been included or not? To my knowledge, the UK is in Europe.\nThe mentioned effort on narrowing the science-policy gap is laudable, but can we expect getting the researchers onboard?\nThe authors mention âtraditionalâ mechanisms around engagement, knowledge transfer, and research assessment, but in particular with respect to the latter, there are only fashions, but no tradition. A tradition only gets established once something is passed on from generation to generation, while we saw substantial changes on shorter time-scales. Notably, the h-index was not invented before 2005.\nThe authors argue that universities are well-positioned to âeducateâ their faculties, but are we facing a question of education? If researchers are motivated, arenât they in need of support rather than incentives?\nChannels to reach policy makers is certainly a relevant point, but looking at social media and news services, the question of quality pops up. Scientists with a large public followership are not necessarily the best suited to speak on a specific topic.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": [
{
"c_id": "8457",
"date": "08 Jul 2022",
"name": "Andrew Kelly",
"role": "Author Response",
"response": "Reponses have been added in-line below the reviewer's comments and are shown in italics. Reviewer's comments: I am having some difficulties with the policy framing of the article, and it does not become obvious what point exactly the authors intend to make. A few statements donât appear to match up. The article touches on many topics, but I feel that none of them are discussed to a sufficient extent. It initially centres around 3 key questions that are to be addressed by a survey of authors, but towards the end it morphs into an essay on developing an environment that supports translating research into policy, which is not much underpinned by the survey data. Consequently, the article feels like two that are loosely connected. Moreover, it sometimes reads like a policy statement and advertisement by Taylor & Francis rather than a research article. This impression is strengthened by the lack of research articles amongst the references in conjunction with the authors not adequately positioning their findings in the context of other research on the topic. We agree that the article needed to be more focused and has been reframed around the results of the author survey, rather than the non-citation value of academic research. The policy framing has been removed. There is a fundamental conflict between the aspiration of âmaximising the capability of research to achieveâ and focusing efforts on addressing urgent needs, which remains unresolved in the article. Throughout, these are conflated and confused. I would consider âcapabilityâ a most relevant keyword in this context. Several references are ill-chosen for supporting the specific point that the authors try to make. Specifically, the Lisbon strategy (reference 1) has the declared aim âto make Europe the most competitive and dynamic knowledge-based economy in the world, capable of sustainable economic growth with more and better jobs and greater social cohesionâ, but it does not include an explicit call for research to have âimpactâ. Moreover, the report of reference 21 states that âPeople are broadly split on whether the UK invests too much in long-term R&D rather than solving issues that matter now (33% agree vs. 35% disagree)â. The link in reference 20 does not work. It is rather unusual for me to defend Michael Gove, but âwe no longer need expertsâ is a (popular) misquotation by omission. He stated: âI think the people of this country have had enough of experts with organisations with acronyms saying that they know what is best and getting it consistently wrong.â, and the latter part of the statement is much relevant for scientists engaging in public debate. It is important to know how to build and maintain trust. Yes, this section was initially reframed to focus on the increased public trust in science/research during the pandemic, but has now been removed owing to the tighter focus and the references have been updated. While the authors elaborate on the term âimpactâ, it remains problematic and likely to be understood in various ways. One could challenge the statement about quantification in economic terms being straightforward, and in particular question whether benefits should be evaluated in such a way. For example, people dying early could be economically beneficial, but would that be societally desired? We agree that the difficulty in qualifying impact itself is part of the challenge, whilst quantification isnât necessarily a good thing. We have reframed around the mobilisation/transfer of knowledge. The âmissionsâ approach in Horizon Europe is somewhat controversial. Notably, the recent 2020 Euroscience Open Forum (ESOF) included a session âDoes science for missions undermine the missions of science?â. Likewise, the authors state that the drive to solve \"the Grand Challenges of our time\" has acquired increased urgency during the COVID-19 pandemic, but one could also argue that prominently reveals a potential flaw of focusing on identified challenges, which is in neglecting those strands of research that are most suitable to provide the basis for the next challenges that we are to encounter (e.g. https://www.statnews.com/2020/02/10/fluctuatingfunding-and-flagging-interest-hurt-coronavirus-research/ and https://www.nytimes.com/2021/04/08/health/coronavirus-mrna-kariko.html) Yes, we agree, although the missions concept is gaining traction worldwide, potentially at the expense of curiosity-driven research. The discursive elements around Horizon Europe has been removed as part of the tightening of the article. The authors refer to citations as the primary currency of success or progress, but it might be worth keeping in mind that it is a widespread myth that this applies universally. In particular, the quoted UK Research Excellence Framework (REF) is not based on citation counts. Moreover, there are significant differences between âimpactâ in the REF and âPathways to impactâ in the context of funding applications to UK Research Councils. Both are distinct from the âPathways to Impact initiativeâ (reference 11). I appreciate the authors mentioning a âchain reactionâ emerging from original research. Could one elaborate on what would determine the value of research? With regard to the role of academic publishing, I note that the International Science Council has recently published an insightful report âOpening the record of science: making scholarly publishing work for science in the digital era.â1 Thank you for sharing the reference. Our experience suggests that this is the case, along with other examples, such as Horizon Europeâs business case, which comments on increased citations comparatively, but this has been modified or removed in the Introduction. The definition of the three main aims of the survey does not specify what group of people âour communitiesâ refers to. I am far less confident than the authors about the respondents being ârepresentativeâ. I would expect that those who just care about their bibliometric profile and other similar performance indicators are not inclined to spend any time responding, which would result in the respondents being more engaged for the scientific community and the wider society. We also agree that survey sampling tends to lead to some degree of self-selection, which may emphasise some biases. However, we were satisfied that the total number of responses across a wide range of journals and the geographical alignment of the respondents with the journalsâ author base allowed us to have reasonable confidence in the representative nature of the results. I found the ânote about error bars and statistical significanceâ almost entirely stating trivialities, whereas the authors do not provide the crucial information of what the quoted âerror barsâ actually refer to and what they mean, which leave me unable to interpret them. The error bars plot the confidence intervals for the percentages shown. If the error bars for two or more countries overlap, we have been cautious about making any substantive conclusions, because they may not be statistically significant, and only clearly statistically significant differences are discussed in our comparisons. There are some substantial weaknesses with the survey questions and the provided answer options. It is somewhat confusing that in some cases respondents were able to pick any number of answers from a list, whereas in others the number of choices was limited. This poses some difficulties for the interpretation of the results and the limitations to answer options should be mentioned clearly in the respective figure and/or captions. It would also be useful if the authors referred to the question numbers. The format of the question was selected according to the purpose of the question and the number of perceived answers. The phrasing of the questions was appropriate for the settings that were used; that is, the questions that presented a limited number of options used wording that emphasised priority, whereas the questions that allowed for an unlimited number of selections used wording that emphasised relevancy. With regards the question that allowed a maximum of three responses, this was labelled clearly within the paper. within the text and chart labels. My main concern with regard to the survey is about Q14 and Q16, which refer to âreal-word problemsâ. I think that it is an unfortunate choice that the authors put these central rather than referring to Q11 in conjunction with Q3 on addressing the question on why researchers undertake the research that they do. While the term âreal-world problemâ carries a polemic tone suggesting that academics might be detached from reality, âdirectly or indirectly contributingâ is remarkably fuzzy. It is not clear to me what Q14 and Q16 are actually able to capture, and I feel that answering with âyesâ, ânoâ, or âDonât knowâ is mostly a matter of interpretation of the question. I could make a case for my research falling into either of these categories, depending on what point of view I assume. In fact, âdonât knowâ appears to be a good option given that for some research the connection to âreal-word problemsâ is not immediately apparent and the connection might only be built in the future. Apparently, a substantial number of respondents chose that option. I also note that Q16 refers to âpriorityâ whereas Q14 does not. I did not see the authors commented on lower numbers for an affirmative response on Q16 as compared to Q14. We used the phrase âreal-world problemsâ as we felt it was commonly used parlance in aggregating topics such as the SDGs, but agree that there is some subjectivity there and there is a need for education of researchers in contextualising their work. We have included a brief section to introduce the terms below the note on error bars. I also wonder how many of the respondents are familiar with what the UN SDGs are, or are willing to look at up before they answer the question. I note that SDG 8 explicitly recognises creativity and innovation as drivers of economic growth, which aligns fundamental research, not directly targeted at specific challenges, with the UN SDGs. Whilst we did not probe the degree of familiarity of the respondents with the UN SDGs, or the scope of individual Goals or Targets, we believe that, especially in the subject areas covered by the survey, it is reasonable to assume that most respondents are broadly familiar with the priorities of the SDGs and that respondents who felt they were not sufficiently familiar with the SDGs to answer the question would have answered âdonât knowâ. Something that puzzles me is that 38% of the respondents did not choose the answer âI find researching these topics interestingâ. Why do they do research that they are not interested in? It would seem to me that the survey reveals another âgapâ than the one the authors claim. A key gap appears to be in how the research actually materialises into something useful, with only about 20% of the survey respondents stated that âinput into policy decision-makingâ or âcontribution to tackling big real-world problems, such as those expressed by the UN SDGsâ was amongst the most important forms of âimpactâ (although they were limited to 3 answers). Thank you for sharing this observation, which has been included in the revision. In contrast, the authors elaborate on the point that respondents ranked formal recognition over making a contribution and state that we risk devaluing the necessary application of original research to addressing our global challenges by prioritising other metrics and outcomes. However, their study does not provide evidence for that. If the research of the respondents is oriented towards âreal-world problemsâ, the underlying motivation is not the relevant issue. Unfortunately, the authors do not elaborate on to what extent âcontribution to the advancement of researchâ is aligned with âcontribution to tackling real-world problemsâ and/or âinput into policy decision-makingâ or rather not. It is the more unfortunate that respondents were restricted to a maximum of 3 answers for Q11 rather than being able to state where each of them ranks in priority. We have added a new Venn diagram (Figure 5), which looks at the overlap of responses to three of the key options from question 11. The percentages are based on the total number of respondents selecting at least one of these three options. We chose to limit the number of responses to Q11 because we felt that, whilst it would have been worthwhile asking respondents to rank all of the answers, it would have been a much-larger undertaking to ask respondents to rank or rate nine answers. Additionally, asking respondents to rank all of the answers would not have allowed them to rank things as equally important/unimportant, or to leave some items unranked. On the question of why authors chose to submit their manuscript to the specific journal, the top chosen answer is pretty much an umbrella category that encompasses more than half of the other answer options, which are more specific on what most ârelevantâ means. The authors should define what they consider âEuropeâ, e.g. if respondents stated that they are located in Turkey, have their answers been included or not? To my knowledge, the UK is in Europe. The mentioned effort on narrowing the science-policy gap is laudable, but can we expect getting the researchers onboard? A mapped list of countries to the regions that were used in the analysis has been added to the FigShare deposit. The authors mention âtraditionalâ mechanisms around engagement, knowledge transfer, and research assessment, but in particular with respect to the latter, there are only fashions, but no tradition. A tradition only gets established once something is passed on from generation to generation, while we saw substantial changes on shorter time-scales. Notably, the h-index was not invented before 2005. The authors argue that universities are well-positioned to âeducateâ their faculties, but are we facing a question of education? If researchers are motivated, arenât they in need of support rather than incentives? Channels to reach policy makers is certainly a relevant point, but looking at social media and news services, the question of quality pops up. Scientists with a large public followership are not necessarily the best suited to speak on a specific topic."
}
]
}
] | 1
|
https://f1000research.com/articles/10-36
|
https://f1000research.com/articles/12-239/v1
|
03 Mar 23
|
{
"type": "Research Article",
"title": "Iron folic acid consumption and anemia prevalence among female adolescents in rural areas: an observational study",
"authors": [
"Apriningsih Apriningsih",
"Rahayu Putri Nopitasari",
"Laily Hanifah",
"Chandrayani Simanjorang",
"Feda Anisah Makkiyah",
"Farwah Hafidah",
"Widayani Wahyuningtyas",
"Rahayu Putri Nopitasari",
"Laily Hanifah",
"Chandrayani Simanjorang",
"Feda Anisah Makkiyah",
"Farwah Hafidah",
"Widayani Wahyuningtyas"
],
"abstract": "Background: Anemia among female adolescents can cause maternal deaths due to bleeding and eclampsia.\n\nAnemia is experienced by over half of billion women of childbearing age (15-49 years) worldwide, and the prevalence increased from 28.5% in 2015 to 29.6% in 2019.\n\nThe maternal deaths in Bogor Regency were mostly caused by bleeding and eclampsia. This study examines the association between individual characteristics, menstrual patterns, nutritional status, and weekly iron folic acid consumption on the anemia status of female adolescents in Sirnagalih, Bogor, West Java. Methods. This is cross-sectional research conducted from December 2021 to February 2022. The study population was female adolescents (10-19 years) and the sample consisted of 78 respondents selected by using a purposive sampling technique. Interviews were conducted using a questionnaire and blood test to collect data. Results: The results of the chi-square analysis showed a significant association between weekly iron folic acid consumption and anemia status of female adolescents (p-value = 0.018 and POR = 0.128 (95% CI: 0.02-0.69)), while other variables did not have a significant association. Conclusions: For female adolescents in rural regions, where the prevalence of anemia is higher than in urban areas, it is necessary to promote iron folic acid supplement consumption. For further research, it is suggested to add other variables that affect anemia status in rural female adolescents such as the availability of another high-protein food, adherence to taking iron folic acid supplements, and to be examined for worm infections.",
"keywords": [
"anemia",
"female adolescent",
"iron-folic acid",
"menstruation pattern",
"nutrition status",
"rural area"
],
"content": "Introduction\n\nThe World Health Organization (WHO) reports that over half a billion women of childbearing age (15-49 years) have anemia.1 Therefore, the United Nations decided to include the prevalence of anemia among pregnant and non-pregnant women as a significant indicator in the 2020 Sustainable Development Goals (SDGs).2 By 2025, the World Health Assembly requested that the prevalence of anemia among women of reproductive age (15-49 years) decrease by 50%; however, this deadline could be extended to 2030.3\n\nThe prevalence of anemia in the world increased from 24.8% in 1993 to 25.4% in 2010, with adolescents as the most significant proportion.4 Anemia prevalence in women of childbearing age worldwide increased in 2015 from 28.5% to 29.9% in 2019.5 An increase in cases of anemia also occurred in female adolescents in Indonesia, from 18.4% in 2013 to 27.2% in 2018.6 A persistent discrepancy between a personâs dietary iron intake and their bodyâs physiological needs results in iron deficiency anemia.7 Female adolescent anemia is a nutritional issue that can seriously affect growth, cognition, work performance, and other areas throughout the reproductive years of life and beyond.8 Pregnancy during adolescence with anemia raises maternal morbidity and mortality rates and the likelihood of unfavorable pregnancy outcomes like stillbirth, low birth weight, and premature birth. It also has a detrimental effect on the infantâs iron status.8 The maternal deaths in Bogor District Regency were caused mainly by bleeding and eclampsia.9 Previous studies explained that female adolescent anemia prevalence can cause maternal death due to bleeding and eclampsia and also low birth weight.10,11\n\nThe factors that influence the incidence of anemia include the level of education, sex, region, breakfast habits, health status, and state of body mass index (BMI) which is included in the underweight category.12 The study results showed that food intake, age, place of residence, and activity type affect hemoglobin levels in a female adolescent who takes iron folic acid supplements.13 Previous studies examined the causes of anemia in female adolescents, consisting of nutritional status, menstrual cycle and length, lack of iron-folate intake, and vitamin B12 as direct factors for anemia in female adolescents.14,15 Other studies have tried to examine indirect causes of anemia such as socio-demography (age, education, economic status).16 In addition, the influence of behavioral factors such as the application of personal hygiene and environmental sanitation and adherence to consuming iron folic acid tablets was also investigated with the incidence of anemia in adolescent girls.17,18\n\nHeavy monthly flow puts female adolescents at risk for iron deficiency anemia, with an incidence of about 9%, and another 15% to 20% of them have iron insufficiency without anemia.19 Adolescent girls who have experienced menstruation are at risk of suffering from anemia due to bleeding during menstruation. This is exacerbated if the dietary intake pattern of iron food sources is significantly less than the recommended adequacy rate. Apriningsih et al.âs 2021 research reported a relationship between the intake of food sources of iron and Hb levels in adolescent girls.20 Intake patterns are influenced by several related factors including socioeconomic status, education, gender, and place of residence.21\n\nWHO and the Indonesian Ministry of Health launched weekly iron folic acid supplementation (WIFAS) program as an effort to prevent and treat anemia in female adolescents.22,23\n\nMost of the WIFAS programs are carried out in schools. However, this program was hampered due to the coronavirus pandemic. There have been several adaptations and innovations that program managers have made to respond to the Ministry of Healthâs policies in dealing with pandemics, such as implementing health protocols, using social media and applications for education and monitoring compliance with IFAS consumption.24 An example is the Ministry of Health of the Republic of Indonesia launching the Ceria application for the prevention of anemia in young women and implementing it in several regions.25 However, this requires facilities and infrastructure such as gadgets and internet networks, and not everyone in rural areas have these, both in developed and developing countries.26,27\n\nA previous study discussed factors related to anemia in general, without focusing on rural areas particularly in Indonesia. According to national health research 2018 data, the proportion of people with anemia is higher in rural areas (25.0%) than in urban areas (22.7%).28 Therefore, this study aims to examine the factors related to anemia in female adolescents in rural areas particularly related to an iron folic acid supplement program.\n\n\nMethods\n\nThe protocol for this study was approved by the Universitas Pembangunan Nasional Veteran Jakarta Health Research Ethics Committee under the number 499/XII/2021/KEPK. The date of approval was 21 December 2021. Verbal consent was provided by participants which the interviewers then noted down. Parents and children gave consent before being involved as research participants; they were not involved as research participants if they did not agree.\n\nThe study was conducted in Sirnagalih Village, Jonggol, Bogor Regency, West Java Province, Indonesia. This village is located in one of the districts and provinces that have the highest maternal mortality rate in Indonesia. The biggest cause of maternal death in Indonesia, including in Bogor, West Java, is bleeding and factors related to the incidence of bleeding including anemia.29\n\n103 potential female adolescent participants were identified by asking village heads and local community health workers. Participants who met the inclusion criteria were asked about their willingness to participate in the study and asked for their parentsâ approval. The approach process was assisted by local community health workers. There were 25 potential participants who are not willing to participate because they have another activity and could not be interviewed at the study time. 78 female adolescents enrolled in the cross-sectional study from December 2021 to January 2022. We used The Lemeshow sample size formula for calculating sample size:\n\nData was collected by interviewing respondents face to face using questionnaires. Examination of hemoglobin levels and interviews were conducted at the village hall of Sirnagalih, Jonggol, Bogor, West Java.\n\nThe independent variables used were the menstrual cycle, duration of menstruation, and consumption of blood-added tablets. The dependent variable (outcome) used was the incidence of anemia in adolescent girls. Confounding variables suspected in this study were age and education level.\n\nThe topics in the questionnaire are menstrual cycle, duration of menstruation, nutrition status, education level and consumption of iron tablets. The researchers created the questionnaire in accordance with the research objectives. Therefore, validity and reliability tests were carried out before being used in the research sample as pilot study. The menstrual cycle is the distance between the start of the last menstrual period and the next menstruation. Menstrual cycle variables were measured using 4 multiple choice questions. Menstrual cycles are categorized into two, standard 21-35 days and abnormal if the menstrual cycle is more than 35 days. Menstruation duration is the duration (day) experienced by a woman during the menstrual process. The menstrual duration variable was measured using 6 multiple choice questions and 1 free text question for confirmation Menstruation duration is categorized into two categories, namely normal (3-8 days) and abnormal (<3 days or >8 days). The variable iron folic acid consumption was measured using 5 multiple choice questions and 1 free text question as supporting information. Respondents are said to be taking Fe tablets if they consume iron tablets regularly every week (according to the program).\n\nQualitative data is collected to add supporting information to the quantitative data. There is one open question that collects qualitative data combined alongside the quantitative questions. The answers given by the respondents were written in the form of a questionnaire.\n\nAnemia status variables were measured using 4 multiple choice questions and measurement of Hb levels to validate respondentsâ answers. Meanwhile, the cyanmethemoglobin method is categorized as anemia (<12 g/dl) and not anemia (â¥12 g/dl). The anemia threshold for women aged 11 years and above is if the concentration or level of hemoglobin in the blood is less than 12 g/dl.31\n\nIndependent variable (age) is categorized based on median value, education level grouping is based on up to junior high school and senior high school or above, nutrition status grouping is based on ideal (Z score -2SD until +1SD) and unideal, menstruation pattern grouping is based on normal and abnormal, and whether or not participants were consuming iron folic acid.\n\nHealth workers carried out data collection via hemoglobin examination to measure respondentsâ Hb levels (Figure 1). This was done by taking a blood sample at the fingertips using an EasyTouch GcHb tool. The quantitative data were analyzed univariately to display the distribution and frequency of the existing variables. We used the chi-square test to examine the association between the variables. Crude OR was obtained in bivariate analysis with α=0.05 and 95% confidence interval (95% CI). All statistical analysis was performed using SPSS Software (25th version, International Business Machines Corp., New York). The multivariate test was not carried out because only one variable met the entry requirements for the multivariate test model. Thus, no interaction test between variables was carried out. There is no missing data in this study.\n\nThe risk of bias that may occur includes selection bias caused by the selection of samples that are not random so that they are not representative. The bias can interfere with the external validity. Another possibility of bias is that there are several other variables that may be associated factors for anemia but were not measured in this study.\n\nQuestionnaire tests were carried out in the month of January 2022 to 50 respondents outside the study population with the same characteristics. Participants were selected by non-probability sampling technique. Based on this test, it was found that the questionnaire was valid with r value= 0.278, while the reliability of the questionnaire was very good (Cronbachâs α = 0.65).\n\n\nResults\n\nBased on information from Sirnagalihâs village head on December 2021, there were 103 people recorded on rural data (female adolescents 10-19 years old, lived in Sirnagalih Village areas, have menstruated). The final number of respondents was 78 female adolescents who were willing to be a respondent; the others were unwilling to participate in this study. This study only applied to female and not male adolescents.\n\nTable 1 presents the characteristics of the respondents. Most participants are older than 15 years old but less than 19 years old (53.8%). Most participantsâ last education level was junior high school level (60.3%). Most participants had standard nutritional status (83.3%), had normal menstrual patterns (59.0%), and did not consume iron folic acid supplements regularly (89.7%).\n\nThese research findings demonstrate that the majority of female adolescents in Sirnagalih village are between 15-19 years old, and that the majority of them have recent education up to junior high school. This is understandable because the compulsory education program in West Java only lasts for 9 years (junior high school level). Even though they live in a village, the average nutritional status of the respondents is included in the good nutrition category based on WHO anthropometry (Z score -2SD to +1SD).\n\nInterestingly, in this village, information was obtained about a mother who was still an adolescent and had anemia. It is allegedly assumed that it is not just one person, but several mothers married young or are classified as teenagers and have anemia, as stated below:\n\nâI got married when I was 17 years old, and some of my friends also got married at a young age, although not manyâ (Respondent X).\n\nThis study showed a relationship between the consumption of iron tablets and anemia in female adolescents (p=0.01, POR=0.13) (see Table 2). In contrast, other variables such as age, education, nutritional status, and menstrual patterns showed no significant relationship (p>0.05).\n\n\nDiscussion\n\nIn this study, the prevalence of anemia among female adolescents in Sirnagalih village was 10.3%. This figure is lower than the national figure (27.2%) and the findings of Sari et al. (2022) in Soreang, West Java (14.3%).28,32 However, it is similar to the prevalence of anemia among adolescents in rural areas in western China (11.7%)33\n\nEarly marriage or marriage before the age of 18 is common in developing nations and is related to a number of social, physical, and health issues.34 Women who marry young typically have a low level of education. Because they have to start caring for a child, they neglect their health. Additionally, early-married women are far more likely to experience domestic abuse.35 The limited availability of nutritious foods, lower socioeconomic status, and lack of access to hygienic sanitation facilities are associated with elevated rates of disease which in turn may be associated with increased risk of anemia in rural areas. Research in Albania shows that the women who lived in rural regions were more vulnerable to being anemic than the women who lived in urban areas.36\n\nMost anemic female adolescents in this study had a senior high school level of education. This finding is in line with a study by Bellizzi et al. (2020) in India, who found that many adolescent girls suffer from anemia with higher education than primary school.37 This is different from the study of Ma et al. (2017) in China, which found that most anemia sufferers in non-pregnant reproductive women had junior high school education.38 This difference is assumed to be because those with junior high school education still get iron-folic acid supplements from their schools. In contrast, those with senior high school education and above do not get them.\n\nIn contrast to the findings from this study, a personâs nutritional condition affects the incidence of anemia.39 A personâs nutritional status (malnutrition) is one of the affectional factors for anemia. The cause of that problem is that young women often eat unhealthy foods like fast food because they ignore their health.31\n\nThey also drink tea or coffee less than an hour after meals, which might interfere with iron absorption.40 Moreover, they often undergo unhealthy diets without an instructor, such as a doctor or nutritionist, affecting their growth and nutritional needs.41\n\nThe menstrual pattern has no significant association with female adolescent anemia. This differs from the meta-analysis study that Endale et al. (2022) conducted. Those studies identified factors associated with female adolescent anemia, such as low dietary diversity (OR: 1.56; 95% CI: 1.05, 2.32), illiterate mothers (OR: 1.45; 95% CI: 1.13, 1.86), households with more than five people (OR: 1.65; 95% CI: 1.14, 2.38), food insecurity in the home (OR: 1.48; 95% CI: 1.21, 1.82), and menstrual blood flow lasting longer than five days (OR: 6.21; 95% CI: 1.67).14\n\nThis study found that a factor significantly related to anemia in young women in rural areas was the consumption of iron folic acid supplements. These results align with the study on Ghana (2021) and Bhutan.42,43 This result reinforces the need for an iron-folic acid supplement program to prevent and treat anemia in female adolescents.\n\nThe supplementation iron-folate program guide by WHO and Ministry of Health of the Republic of Indonesia should be sustainable and monitored for its effectiveness.1,23,44 Its effectiveness includes the issue of adherence to drinking iron folic acid for female adolescents and the level of school readiness as the spearhead of the programâs success. The pandemic period that has hampered health programs, including the WIFAS school-based program, should give rise to innovations such as using internet-based social media for education and promoting adolescent health, including anemia. However, internet-based social media is very limited in its application in rural areas, where internet facilities are still limited.\n\nThis study has limitations in the number of samples, types of causative factors, and study design by using a cross-sectional design. A cross-sectional design was appropriate in this study because it was effective in observing the phenomenon and causes at the same time. Therefore, it is recommended for further research to increase the number of samples, add causal factors, and use a more robust study design such as a cohort or experiment.\n\n\nConclusions\n\nIron folic acid supplement consumption factor is associated with anemia in female adolescents in rural areas. Therefore, it is necessary to organize an iron folate supplementation program in school-based to reach female adolescents who do not attend school in rural areas.",
"appendix": "Data availability\n\nFigshare: Complete Raw Data, https://doi.org/10.6084/m9.figshare.22010306.v1. 45\n\nFigshare: Kuesioner anemia inggris.docx, https://doi.org/10.6084/m9.figshare.21922059.v1. 46\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors would like to thank the head of Sinargalih Village for granting permission, as well as the leader of the youth organization and cadre for collaborating with the research. Thank you also to all female adolescents who participated in our study.\n\n\nReferences\n\nWorld Health Organization: The Global Prevalence of Anaemia in 2011. WHO Report. 2015; 48. Reference Source\n\nUNGA: Global Indicator Framework for the Sustainable Development Goals and Targets of the 2030 Agenda for Sustainable Development 2020. Reference Source\n\nYoung MF, Oaks BM, Rogers HP, et al.: Maternal Anemia and High Hemoglobin Concentrations and Association with Adverse Maternal and Infant Health Outcomes: An Updated Global Systematic Review and Meta-Analysis. preprint; In Review 2022. Publisher Full Text\n\nMarfuah D, Pertiwi D, Kusudaryati D: Effectiveness of Nutrition Education on Improving Iron Intake in Young Women. PROFESI. 2016; 14(1): 5. Publisher Full Text\n\nWorld Health Organization: Anaemia in Women and Children. THE GLOBAL HEALTH OBSERVATORY. World Health Organization; 2021. (accessed 2022-02-03). Reference Source\n\nMinistry of Health RI: Health Research and Development Agency. National Report of Basic Health Research 2018. Jakarta: 2019; pp. 614â614.\n\nAbbaspour Nazanin RH, Kelishadi R: Review on Iron and Its Importance for Human Health. J. Res. Med. Sci. 2014; 819(2): 164â174.\n\nMengistu G, Azage M, Gutema H: Iron Deficiency Anemia among In-School Adolescent Girls in Rural Area of Bahir Dar City Administration, North West Ethiopia. Anemia. 2019; 2019: 1â8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBogor City Health Office: Health Profile of Bogor Regency. West Java: Bogor; 2019.\n\nMoraes AN, Likwa RN, Nzala SH: A Retrospective Analysis of Adverse Obstetric and Perinatal Outcomes in Adolescent Pregnancy: The Case of Luapula Province, Zambia. Matern. Health Neonatol. Perinatol. 2018; 4(1): 20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaz U: Comparison of Obstetric Outcome in Terms of the Risk of Low Birth Weight, Preterm Delivery, Cesarean Section Rate and Anemia in Primigravid Adolescents and Older Primigravida.2014; 24: 4.\n\nSayed SF, Nagarajan S: Haemoglobin Status to Determine Nutritional Anaemia and Its Association with Breakfast Skipping and BMI among Nursing Undergraduates of Farasan Island, KSA. J. Nutr. Sci. 2022; 11: e36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIllahi D: Overview of Hemoglobin Levels in Young Women Who Consumed Iron Supplementation/Fe. Journal of Ners and Midwifery. 2015; 2(1): 020â029. Publisher Full Text\n\nEndale F, Woldeyohannes D, Belayneh F, et al.: Menstrual Abnormality, Maternal Illiteracy, and Household Factors as Main Predictors of Anemia among Adolescent Girls in Ethiopia: Systematic Review and Meta-Analysis. Womenâs Health (Lond. Engl.). 2022; 18: 174550572211293. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThomas D, Chandra J, Sharma S, et al.: Determinants of Nutritional Anemia in Adolescents. Indian Pediatr. 2015; 52(10): 867â869. Publisher Full Text\n\nFatusi AO, Bello B: Social Determinants of Health in Adolescents and Young Womenâs Health and Nutrition: Current Evidence. Nestlé Nutrition Institute Workshop Series. Vol. 80. . Bhutta ZA, Makrides M, Prentice AM, editors. S. Karger AG; 2015; pp. 61â69. Publisher Full Text\n\nDhillon PK, Kumar B, Verma HK: Prevalence of Anemia in View of Socio-Demographic and Health Status of Adolescent Girls Enrolled in Government School at Border-Belt of Indian Punjab. Ecol. Food Nutr. 2021; 60(2): 198â211. PubMed Abstract | Publisher Full Text\n\nSingh M, Rajoura O, Honnakamble R: Anemia-Related Knowledge, Attitude, and Practices in Adolescent Schoolgirls of Delhi: A Cross-Sectional Study. Int. J. Health Allied sci. 2021; 8: 144â148.\n\nFentie K, Wakayo T, Gizaw G: Prevalence of Anemia and Associated Factors among Secondary School Adolescent Girls in Jimma Town, Oromia Regional State, Southwest Ethiopia. Anemia. 2020; 2020: 1â11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nApriningsih A, Dwiriani CM, Madanijah S, et al.: High School Female Studentsâ Adherence to Iron Folic Acid Supplement Consumptions and Hemoglobin Improvement. Southeast Asian J. Trop. Med. Public Health. 2021; 52(Supplement 1).\n\nBeck KL, Jones B, Ullah I, et al.: Associations between Dietary Patterns, Socio-Demographic Factors and Anthropometric Measurements in Adult New Zealanders: An Analysis of Data from the 2008/09 New Zealand Adult Nutrition Survey. Eur. J. Nutr. 2018; 57(4): 1421â1433. PubMed Abstract | Publisher Full Text\n\nApriani Mirza AS: Adolescent Compliance on Iron Tablet Consumption: A Systematic Review. Adv. Sci. Lett. 2018; 24(9): 6371â6375. Publisher Full Text\n\nApriningsih, Madanijah S, Dwiriani CM, et al.: Determinant of High School Girl Adolescentâadherence to Consume Iron Folic Acid Supplementation in Kota Depok. J. Nutr. Sci. Vitaminol. 2020; 66: S369âS375. PubMed Abstract | Publisher Full Text\n\nSoewondo P, Sakti GMK, Irawati DO, et al.: Portrait of Adaptation and Innovation of Nutrition Services during the Covid-19 Pandemic: Case Studies in 8 Regencies/Cities in Indonesia. Annual Scientific Forum. 2020; pp. 25â26.\n\nJayadi YI, Palangkei ASIA, Warahmah JF: Evaluations Of Iron Tablets Supplementation Program For Young Women In The Binamu City Health Center Area. HTJ. 2021; 7(3): 168â175. Publisher Full Text\n\nCurtis ME, Clingan SE, Guo H, et al.: Disparities in Digital Access among American Rural and Urban Households and Implications for Telemedicine-based Services. J. Rural. Health. 2022; 38(3): 512â518. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLopez-Sintas J, Lamberti G, Sukphan J: The Social Structuring of the Digital Gap in a Developing Country. The Impact of Computer and Internet Access Opportunities on Internet Use in Thailand. Technol. Soc. 2020; 63: 101433. Publisher Full Text\n\nBalitbangkes: Basic Health Research 2018. Health Research and Development Agency. 2018; pp. 198â198.\n\nAstari RY, Sandela D, Elvira G: Overview Of Maternal Mortality In Majalengka Regency In 2015 (Qualitative Study). MJ. 2018; 3(1): 69. Publisher Full Text\n\nWeliyati W, Riyanto R: Factor Related to Female Adolescent Anemia at SMAN Kota Metro. Sai Wawai Metro Health Journal. 2012.\n\nMunt AE, Partridge SR, Allman-Farinelli M: The Barriers and Enablers of Healthy Eating among Young Adults: A Missing Piece of the Obesity Puzzle: A Scoping Review: Barriers and Enablers of Healthy Eating. Obes. Rev. 2017; 18(1): 1â17. PubMed Abstract | Publisher Full Text\n\nNovita Sari H, Maryani K, Rusdiyani I: Eearly Childhood Nutrition Intake Patterns During The COVID-19 Pandemic.2022; 7 (1): 51â64.\n\nZhu Z, Sudfeld CR, Cheng Y, et al.: Anemia and Associated Factors among Adolescent Girls and Boys at 10â14âYears in Rural Western China. BMC Public Health. 2021; 21(1): 218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamed A, Yousef F: Prevalence, Health and Social Hazards, and Attitude toward Early Marriage in Ever-Married Women, Sohag, Upper Egypt. EPX. 2017; 92(4): 228â234. PubMed Abstract | Publisher Full Text\n\nTiruneh FN, Tenagashaw MW, Asres DT, et al.: Associations of Early Marriage and Early Childbearing with Anemia among Adolescent Girls in Ethiopia: A Multilevel Analysis of Nationwide Survey. Arch. Public Health. 2021; 79(1): 91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTalukder A, Paul N, Khan ZI, et al.: Risk Factors Associated with Anemia among Women of Reproductive Age (15â49) in Albania: A Quantile Regression Analysis. Clin. Epidemiology Glob. Health. 2022; 13: 100948. Publisher Full Text\n\nBellizzi S, Pichierri G, Panu Napodano CM, et al.: Iron Deficiency Anaemia and Low BMI among Adolescent Girls in India: The Transition from 2005 to 2015. Public Health Nutr. 2021; 24(7): 1577â1582. PubMed Abstract | Publisher Full Text\n\nMa Q, Zhang S, Liu J, et al.: Study on the Prevalence of Severe Anemia among Non-Pregnant Women of Reproductive Age in Rural China: A Large Population-Based Cross-Sectional Study. Nutrients. 2017; 9(12): 1298. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChapparo GM: Suchdev PS. Anemia Epidemiology, Pathophysiology, and Etiology in Low- and Middle-Income Countries. Ann. N. Y. Acad. Sci.\n\nNazanin A, Richard Hurrell RK: Review on Iron and Its Importance for Human Health. J. Res. Med. Sci. 2014; 819(2): 164â174.\n\nDeivita Y, Syafruddin S, Andi Nilawati U, et al.: Overview of Anemia; Risk Factors and Solution Offering. Gac. Sanit. 2021; 35: S235âS241. Publisher Full Text\n\nCampbell RK, Aguayo VM, Kang Y, et al.: Epidemiology of Anaemia in Children, Adolescent Girls, and Women in Bhutan. Matern. Child Nutr. 2018; 14(S4). Publisher Full Text\n\nGosdin L, Sharma AJ, Tripp K, et al.: A School-Based Weekly Iron and Folic Acid Supplementation Program Effectively Reduces Anemia in a Prospective Cohort of Ghanaian Adolescent Girls. J. Nutr. 2021; 151(6): 1646â1655. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinistry of Health: Guidelines for the Prevention and Control of Anemia in Young Women and Women of Childbearing Age (WUS).2016; p. 14.\n\nApriningsih A, Nopitasari R, Hanifah L, et al.: Complete Raw Data. Dataset. figshare. 2023. Publisher Full Text\n\nApriningsih A, Hanifah L, Makkiyah F, et al.: Kuesioner anemia inggris.docx. Dataset. figshare. 2023. Publisher Full Text"
}
|
[
{
"id": "219669",
"date": "15 Nov 2023",
"name": "Ambarish Dutta",
"expertise": [
"Reviewer Expertise Epidemiology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA study with 10% anemia prevalence (only 10 anemic among 78) at the population level and with 78 respondents only (that too not a probability sample but purposively chosen) has hardly any power. This is manifest by the OR of the principal explanatory variable 0.02-0.69 having extremely wide 95% Confidence Interval which evokes no confidence in the results and the conclusions drawn from the results.\nMoreover the anticipated frequency of anemia in two groups IFAS consumers and non-consumers was based on only one study which was weak itself and these frequencies were used to calculate the sample size which is flawed.\nThe study should be rejected\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "175206",
"date": "15 Nov 2023",
"name": "Pratap Chandra Mohanty",
"expertise": [
"Reviewer Expertise My specialisation is in the area of Health Economics with a focus on nutrition",
"public health challenges"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe theoretical constructs are not fully justified to take this research further. The innovation in this study is not clearly spelled. The approaches to reach the conclusions are incomplete. The sample size is also very small, and the study area does not represent the countries' rural areas since the method is purposive and stuck to the vulnerable areas only.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-239
|
https://f1000research.com/articles/11-1254/v1
|
04 Nov 22
|
{
"type": "Brief Report",
"title": "The number of neutral mutants in an expanding Luria-DelbrÌck population is approximately Fréchet",
"authors": [
"Steven A. Frank"
],
"abstract": "Background: A growing population of cells accumulates mutations. A single mutation early in the growth process carries forward to all descendant cells, causing the final population to have a lot of mutant cells. When the first mutation happens later in growth, the final population typically has fewer mutants. The number of mutant cells in the final population follows the Luria-DelbrÌck distribution. The mathematical form of the distribution is known only from its probability generating function. For larger populations of cells, one typically uses computer simulations to estimate the distribution. Methods: This article searches for a simple approximation of the Luria-DelbrÌck distribution, with an explicit mathematical form that can be used easily in calculations. Results: The Fréchet distribution provides a good approximation for the Luria-DelbrÌck distribution for neutral mutations, which do not cause a growth rate change relative to the original cells. Conclusions: The Fréchet distribution apparently provides a good match through its description of extreme value problems for multiplicative processes such as exponential growth.",
"keywords": [
"Population genetics",
"probability distributions",
"extreme value distributions"
],
"content": "\n\nSuppose a single cell expands exponentially to a population of size N, with a mutation rate of u per cell division. The number of mutant cells, m, in the final population depends on the number of mutations that occur and when those mutations occur. For example, a single mutation in the final round of cell division is limited to one cell. By contrast, a single mutation transmitted to one of the daughters in the first cellular division may occur in approximately one-half of the final population.\n\nThe distribution of the number mutants, m, is known as the LuriaâDelbrÃŒck distribution1. That distribution is widely used to estimate the mutation rate. The distribution also arises when studying the amount of mutational mosaicism within multicellular individuals2â4.\n\nCurrently, for experiments with a small number of mutational events, one typically calculates the distribution with a probability generating function5,6. However, that approach becomes numerically inaccurate for larger numbers of mutational events, in which case the distribution is calculated by computer simulation.\n\nThis article shows that the Fréchet distribution provides a good approximation for the number of neutral mutants. In particular, the probability that the number of mutants, m, is less than z is approximately\n\nin which exp(z) = ez is the exponential function. The probability of being in the upper tail, m > z, is 1 â F(z). The three parameters set the shape, α, the scale, s, and the minimum value, β, such that z,m > β.\n\nThis form of the Fréchet distribution has three parameters. I found that the following parameterization matches closely the LuriaâDelbrÃŒck process for neutral mutations\n\nin which e is the base of the natural logarithm. This parameterization depends on the single parameter, Nu, the final population size times the mutation rate.\n\nFigure 1 shows the good fit. Two aspects of mismatch occur. First, the number of mutants is discrete, whereas the Fréchet is continuous. As Nu declines to one, significant amounts of probability mass concentrate at particular mutant number values, causing discrepancy between the distributions. Nonetheless, the Fréchet remains a good approximation.\n\nEach population begins with one cell and grows to N cells. Mutation occurs at rate u. Blue curves show the distribution from a computer simulation using the simu.cultures command of the R package rSalvador7. Orange curves show the Fréchet distribution in Equation 1. In rSalvador, I used sample sizes of 106 or 107, values of Nu varying as shown above the plots, and values of N ranging from 106 to 1010. The Julia software code to produce this figure is available from Zenodo8. The input data for calculating the empirical LuriaâDelbrÃŒck CDF is also available from Zenodo9.\n\nSecond, the lower tail of the LuriaâDelbrÃŒck process spreads to lower values than the Fréchet. One can see this mismatch most clearly in the figure for Nu ⥠100.\n\nThis mismatch may occur because the LuriaâDelbrÃŒck process transitions from a highly stochastic process in earlier cellular generations to a nearly deterministic accumulation of mutations in later cellular generations, when the larger population size reduces the coefficient of variation in the number of new mutations. The Fréchet applies most closely to the earlier generations for the following reasons.\n\nIn an expanding population, the earliest mutation strongly influences the final number of mutants. An early mutant carries forward to all descendant cells in an expanding mutant clone. If we start with the final cells and then look back through the cellular generations toward the original progenitor, the mutation with the most extreme time from the end toward the beginning tends to dominate the final mutant number.\n\nThe extreme value of a temporal extent often has a Gumbel distribution. In this case, once the mutation arises, it increases multiplicatively by cell division to affect the final mutation count. Substituting the extreme Gumbel time for its multiplicative consequence provides a common way to observe a Fréchet probability pattern.\n\nPrior mathematical work also supports the Fréchet approximation. Kessler and Levine10 showed that the LuriaâDelbrÃŒck distribution converges to a Landau distribution for large Nu, in which the Landau distribution is a special case of the Lévy α-stable distribution. However, the Landau distribution does not have a closed-form expression for its probability or cumulative distribution functions.\n\nSeparately, Simon11 showed the close match between the Lévy α-stable distribution and the Fréchet distribution. That match of a Lévy distribution to the Fréchet distribution had not previously been associated with the LuriaâDelbrÃŒck distribution. The Fréchet parameterization in this article provides a simple expression that can be used to develop further theory and applications of the LuriaâDelbrÃŒck process.",
"appendix": "Data availability\n\nThe input data for calculating the empirical LuriaâDelbrÃŒck CDF:\n\nZenodo: Empirical CDF for LuriaâDelbrÃŒck distribution from rSalvador package. https://doi.org/10.5281/zenodo.70756559.\n\n\nReferences\n\nZheng Q: Progress of a half century in the study of the LuriaâDelbrÃŒck distribution. Math Biosci. 1999; 162(1â2): 1â32. PubMed Abstract | Publisher Full Text\n\nOtto SP, Hastings IM: Mutation and selection within the individual. Genetica. 1998; 102â103(1â6): 507â524. PubMed Abstract | Publisher Full Text\n\nFrank SA: Somatic mosaicism and cancer: inference based on a conditional LuriaâDelbrÃŒck distribution. J Theor Biol. 2003; 223(4): 405â412. PubMed Abstract | Publisher Full Text\n\nIwasa Y, Nowak MA, Michor F: Evolution of resistance during clonal expansion. Genetics. 2006; 172(4): 2557â2566. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMa WT, Sandri GH, Sarkar S: Analysis of the LuriaâDelbrÃŒck distribution using discrete convolution powers. J Appl Probab. 1992; 29(2): 255â267. Publisher Full Text\n\nZheng Q: Estimation of rates of non-neutral mutations when bacteria are exposed to subinhibitory levels of antibiotics. Bull Math Biol. 2022; 84(11): 131. PubMed Abstract | Publisher Full Text\n\nZheng Q: rSalvador: an R package for the fluctuation experiment. G3 (Bethesda). 2017; 7(12): 3849â3856. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrank SA: evolbio/FrechetLD: F1000 (1.0.1). [Software], Zenodo. 2022. http://www.doi.org/10.5281/zenodo.7255050\n\nFrank SA: Empirical CDF for LuriaâDelbrÃŒck distribution from rSalvador package (1.0.0). [Dataset]. Zenodo. 2022. http://www.doi.org/10.5281/zenodo.7075656\n\nKessler DA, Levine H: Large population solution of the stochastic LuriaâDelbrÃŒck evolution model. Proc Natl Acad Sci U S A. 2013; 110(29): 11682â11687. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimon T: Comparing Fréchet and positive stable laws. Electron J Probab. 2014; 19: 1â25. Publisher Full Text"
}
|
[
{
"id": "155112",
"date": "23 Nov 2022",
"name": "Qi Zheng",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn his brief report the author presents an interesting approximation of the Luria-Delbruck distribution, which microbiologists use to help determine microbial mutation rates in the laboratory. Specifically, equation (1) in the brief report is an approximation of the cumulative probability. If  denotes the probability of  mutants, the author implicitly defines the cumulative probability\n\nas .\n\nThe author's key finding is that , where  is defined by equation (1) in the brief report. Note that the approximation in (1) is valid for any . However, as pointed out by the author, the approximation works well only for values of  that are noticeably larger than . I have conducted a number of computer experiments and confirmed the numerical results in the brief report. The approximation is theoretically interesting, and it may stimulate further theoretical developments. Thus, the paper merits indexing.\nI have a minor comment. There appears to be a typo in equation (1) in the brief report. If Prob() is changed to Prob(), the correlative change in the definition of  will make  conform to the accepted definition of the cumulative probability. (That is, ) More importantly, this may make the approximation more accurate for small . Consider the case\n\n(The symbol  here is the same as the symbol  in the brief report). Table 1 shows results obtained by using the revised definition, while Table 1A shows corresponding results obtained by using the original definition. In both tables, \"error\" refers to the following quantity:\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9363",
"date": "21 Feb 2023",
"name": "Steven Frank",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Thank you for the careful reading. With regard to the comment about m < z versus m <= z, the calculations to make figure 1 used m <= z for the empirical distribution, as recommended by the reviewer. For the theoretical continuous Frechet the numerical values are the same for the two cases. However, I agree that the notation in the original version of the manuscript is misleading. I will post a revised version that uses m <= z, as recommended."
}
]
},
{
"id": "162863",
"date": "20 Feb 2023",
"name": "Pavol Bokes",
"expertise": [
"Reviewer Expertise Mathematical biology",
"stochastic modelling",
"gene expression",
"differential equations"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper compares the empirical (simulational) Luria-Delbruck mutant-number distribution to a Frechet distribution. The advantage of the Frechet distribution over other known approximations, e.g. the skewed alpha-stable distributions, is that it possesses a closed-form cumulative distribution function (cdf), see Equation (1). The parameters alpha, s, and beta of the Frechet distribution are set by the author as specific functions of the population-wide mutation rate N*u. Figure 1 visually demonstrates a solid agreement between the Frechet distribution and the empirical Luria-Delbruck distribution.\nSimilarly to right-skewed alpha-stable distributions, the Frechet distribution has a heavy right tail and a light left tail (for z < beta the density is zero). It follows from (1) that the complementary cdf decays as 1/z^alpha, where alpha has been set by the author to exp(1)/2. In addition to the log-linear plots of Figure 1, it would be interesting to look closer at the power laws of the theoretical and empirical complementary cdfs e.g. using a log-log plot.\nOverall, the paper is well written, presents sound ideas, and develops an interesting approximation to the Luria-Delbruck mutant-number distribution.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9362",
"date": "21 Feb 2023",
"name": "Steven Frank",
"role": "Author Response F1000Research Advisory Board Member",
"response": "Thank you, I appreciate these comments. I agree that looking closely at the upper tail in a log-log plot would provide additional insight about the frequency of rare but potentially important events. However, to achieve a good computational estimate for the true cdf of the assumed process would require some new analyses to obtain precise estimates of the numerical error of the computation. That error could potentially be significant for the very rare upper tail events that would be the focus of such analysis. As applications arise that require a good estimate of the upper tail, the analyses and calculations would be a worthwhile new project."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1254
|
https://f1000research.com/articles/12-236/v1
|
03 Mar 23
|
{
"type": "Software Tool Article",
"title": "Graphie: A network-based visual interface for the UK's primary legislation",
"authors": [
"Evan Tzanis",
"Pierpaolo Vivo",
"Yanik-Pascal Förster",
"Luca Gamberi",
"Alessia Annibale",
"Pierpaolo Vivo",
"Yanik-Pascal Förster",
"Luca Gamberi",
"Alessia Annibale"
],
"abstract": "Background: legislation.gov.uk is a platform that enables users to explore and navigate the many sections of the UKâs legal corpus through its well-designed searching and browsing features. However, there is room for improvement as it lacks the ability to easily move between related sections or Acts and only presents a text-only rendering of provisions. With Graphie, our novel navigational tool (graphie.quantlaw.co.uk), we aim to address this limitation by presenting alternative visualizations of legal documents using both text and graphs. Methods: The building block of Graphie is Sofia, an offline data pipeline designed to support different data visualizations by parsing and modelling data provided by legislation.gov.uk in open access form. Results: Graphie provides a network representation of the hierarchical structure of an Act of Parliament, which is typically organized in a tree-like fashion according to the content and information contained in each sub-branch. Nodes in Graphie represent sections of an Act (or individual provisions), while links embody the hierarchical connections between them. The legal map provided by Graphie is easily navigable by hovering on nodes, which are also color-coded and numbered to provide easily accessible information about the underlying content. The full textual content of each node is also available on a dedicated hyperlinked canvas. Conclusions: While we focus on the Housing Act 2004 for illustrative purposes, our platform is scalable, versatile, and provides users with a unified toolbox to visualize and explore the UK legal corpus in a fast and user-friendly way.",
"keywords": [
"legal data science",
"legislation",
"data pipelines",
"network interfaces",
"visualization of legal texts",
"user interface"
],
"content": "1. Introduction\n\nThe volume of the UKâs primary legislation keeps growing at a very fast pace. According to a rough estimate there are at least 176,890 [1] Public and General Acts currently in force in the UK â the exact number is not known â and an average of 30 new Public Acts are produced every year.1 New legislation [2] documents are regularly uploaded to the UKâs Legislation web platform (legislation.gov.uk2), managed by The National Archives [3] (TNA) on behalf of HM Government.\n\nUsers may reach the legislation.gov.uk webpage while looking for a specific Act or provision on standard search engines. Others may use the platform as part of their daily job. The legislation.gov.uk website has been carefully designed and is maintained to cater for the needs of a diverse pool of stakeholders. It is built on clear principles and offers a number of essential features: first, users can keyword-query the database, and are offered an easy-to-use set of navigational links for browsing through different corners of the UK legislation. Secondly, legislation data are open-source and fully accessible via an API [4]. All API legal documents are held in XML format under a well-defined and concise set of persistent URIs [5]. Thanks to this API technology and to TNAâs open-access philosophy, the legislation data can also be connected and streamlined across other data sets and applications, such as for instance Westlaw,3 a leading commercial legal research platform. In addition, the legislation.gov.uk platform enables users to enjoy the textual version of a whole Act â or a section/paragraph thereof â in HTML or in PDF formats. Acts are made available in both their original (as enacted) or revised (current) versions, and for those Acts with revisions, a detailed timeline highlighting any editing changes to legal documents over time is also provided (see Figure 1).\n\nThe default visualization offering includes (i) plain text of the provision (with limited hyperlinking); (ii) a time-slider to access different versions of the provision; (iii), a choice between the latest version, or version as originally enacted.\n\nThe standard set by TNA in terms of offering a digital and navigable version of essentially the entire corpus of UK legislation is high and very competitive on the world stage. However, the lack of easy âhoppingâ capabilities between items and provisions that should be naturally linked together, as well as its focus on a text-only rendering of provisions leaves room for some improvement.\n\nAs for academic papers, reading and understanding legislation requires concentration and time, and the ability to efficiently âfollow the leadsâ between different provisions of the same Act â or between different Acts that have a bearing on the same matter. Consider again section 194 of the Housing Act 2004 as our main example, which is highly connected [6] with other sections from different Acts of the UKâs Statute Book. To fully understand the content and implications of section 194, the reader is expected to visit and read the sections of these other statutes referenced there first, and then hop onto the sections/provisions that these other sections might refer to, and to repeat this hopping routine exhaustively, covering all possible linkages between sections/provisions/statutes. Using a text-based visualization interface with limited hyperlinking capabilities such as that provided by Ref. 2 makes these tasks time-consuming and inefficient for long and highly interconnected sections.\n\nThus, there is a need for improved tools and visualizations to help both occasional and professional users manage potentially demanding explorations into legal documents. This is exactly the aim of Graphie [7],26 which provides a different and more attractive palette of network visualization tools (see an example in Figure 2) that may prove useful for law researchers and practitioners, as well as for the general public.\n\nOn the left, the web of provisions joined by a link whenever two of them can be reached in one-hop from one another. Nodes are color-coded (and the shape of the node marker can be changed too) to reflect â in this particular case â which Part of the Act each node belongs to. Hovering with the mouse over each node retrieves the textual content embedded in the node (on the right). Moving from each provision to its âneighborsâ no longer requires refreshing or clicking on hyperlinks, but simply wandering around with the mouse over the nodes of interest.\n\nBefore describing the main technical features and capabilities of Graphie, we put our enterprise in the wider context of Legal Map systems and similar initiatives, highlighting overlaps and differences.\n\nRelated work\n\nThe philosophy and technical construction behind Graphie do not write on a blank slate. The concept of a Legal Map system and its theoretical framework were already introduced in Ref. 4. Legal Maps are multi-layered systems offering an end-user experience similar to the user interfaces provided by geographic navigation systems, such as Google Maps [8]. From a mathematical perspective, a Legal Map is a directed multi-graph (network) that represents all the structural elements of a legal source. Reference 5 provides an illustration of a Legal Map network, which quantifies the legal sources of the European Union. Legal Map visualizations could unlock dependencies between legal entities that may be difficult to extract otherwise, from identifying the most âimportantâ nodes in a network, to clustering nodes according to a given notion of similarity.\n\nThe use of tools from complexity science and network theory to analyze and represent legal texts has a relatively short but already fruitful history: arguably, the newly minted âPhysics of the Lawâ field6 will play the same role to Law that Econophysics has to Economics,7 and Mathematical Biology to Biology (e.g. Ref. 8) in terms of cross-fertilization of ideas between distinct domains. An extensive graph-theoretic approach to the EU legislation network is given in Ref. 5. In Ref. 9 the tree-hierarchical network of the U.S. Code is examined by considering several scoring and ranking metrics. Reference 10 builds a hierarchical model of information (distributed on the nodes of a tree) and defines a notion of âstructural complexityâ on the basis of the average time a random reader takes to retrieve some piece of information planted in the leaves.\n\nThe authors of Ref. 11, prior to applying a network-driven analysis against the statutes and regulations in the United States and Germany, perform several pre-processing steps on their underlying raw data. It has been noted in Ref. 12 and in Ref. 13 that legal documents often lack sufficient metadata and are presented as plain text, creating difficulties for their application in legal informatics. We undertake a similar data preparation exercise against our raw data in Section 2.\n\nNetwork-based representations of âinformationâ do exist in other contexts, for instance academic papers. Scholarly archiving systems (Semantic Scholar [9], PubMed [10], Arxiv [11]) use state-of-the-art AI and API engineering that make the processing of scientific documents easier. This enables the development of applications such as Connected Papers [12]. Connected Papers is a network-driven citation analysis tool for enabling end users to explore relevant academic papers. The tool facilitates collecting and analyzing academic references from the chosen archiving system. Network visualizations in Connected Papers are developed using D3.js [13], a well-established JavaScript library for producing bespoke and interactive visualizations. With Graphie, we aim to develop a similar tool, using the same front end technology, but tailored to a different â and arguably less malleable â type of raw data.14 Indeed, the XML versions of the legal texts provided by Ref. 2âs API (footnote 4) have a complex sub-structure, which is markedly different from the short-text nature (say, titles and abstract) typical of academic papers handled by citation APIs. Thus, Graphie faces the extra challenge of having to parse and build a unified model of long and intricate legal documents starting from their (XML) raw representation.\n\nComing back to the legal platforms field, LAWSampo13 is an example of a modern legal semantic web portal in the context of the Finnish Legislation. LAWSampo is built according to the FAIR15 principles of the Sampo Model16 and the SAMPO-UI,17 a full-stack Javascript framework. LAWSampoâs architecture clearly separates the user interface (SAMPO-UI) from the underlying Linked Data service via a SPARQL API [14]. Software developers or legal analysts could use SPARQL endpoints and query LAWSampoâs data service for their own Python or R applications (say network visualizations), using Jupyter notebooks. SAMPO-UI supports network visualizations by including a Cytoscape.js [15] based component, already demonstrated in a few portal instances of the Sampo model, existing in LetterSampo [16] and in AcademySampo [17]. In Graphie, we aim to develop the âVisualizationâ feature, also mentioned in Ref. 18, where sections or provisions of one Act are represented as nodes, and their âconnectionsâ along the information hierarchy as edges. Each node is endowed with its own primary XML schema reference (described below).\n\nTo facilitate the web development of LAWSampo, the authors in Ref. 13 completed a specific data exercise by initially transforming legal documents hosted on Finlexâs Data Bank server Finlex into a Linked Open Data (LOD) repository, named Semantic Finlex.19 Consequently, Semantic Finlexâs data were converted into a data format compatible with LAWSampoâs semantic portal. While we do not use the concept of LOD in our work, we took a similar data pre-processing exercise in Graphie. Prior to any visualization, in Section 2 we illustrate how XML raw data from Ref. 2 are parsed and then checked against data quality indicators. The Graphie Data Model is not strictly following the FAIR principles, as it is not published as an open ontology.\n\n\n2. Methods: The Cross-Act pipeline, Sofia\n\nGraphieâs aim is to tame the complexity of long and intricate legal texts by departing from the traditional âtext+hyperlinksâ philosophy adopted by most digital archives, in favor of a more holistic, network-based representation of the underlying information content. We achieve this goal by defining the following multi-phase pipeline (see Figure 3):\n\n1. We codify and represent one Actâs hierarchical structure and its textual content using a programming language (in our case, Python). In Section 2.1 (Data modelling), a Python object is accordingly declared, henceforth named: Graphie Object (or Graphieâs Data model).\n\n2. We parse the raw XML document of one specific Act using the XML parser defined in Section 2.2 (Parser). Ingested data feed an instance of the Graphie Object, defined in phase 1.\n\n3. We undertake several data integrity procedures against the parsed textual elements of the Graphie object from phase 2 to ensure their quality and completeness. (Data integrity)\n\n4. We convert, using Python, the obtained instance of the Graphie Object from phase 3 into specific JSON files or HTML components. (Transformation service)\n\n5. We use the JSON files and the HTML components from phase 4 for finally feeding the underlying network libraries and certain HTML parts of our platform. (Visualizations)\n\nTo implement this pipeline, we apply a multi-phase procedure that includes the following: data modelling, parser, data integrity, transformation service and visualizations. Data integrity routines are performed and hosted outside the mentioned Jupyter page.\n\nWe designed this pipeline with data analysts in mind. Thus, Sofia25 is implemented as a Jupyter notebook which offers a streamlined experience for capturing and processing UK Legislation XML documents. As we discuss in Section 2.3, data checking tools and activities are hosted outside the aforementioned Jupyter notebook and based on the outcome, the XML parser component of phase 2 is expected to be adjusted accordingly. For this reason, Sofia is an example of an offline pipeline in which we carry out data integrity checks on outputs of phase 2 and phase 4. In the following subsections we describe in detail the different phases of our pipeline, before discussing future work in Section 4.\n\nPrior to any graph visualization, data modelling of the original data is required. In our case, we need to map elements of the UK Legislation XML files into a target data model, which is closer to our final network representation. This requires identifying: which elements from the original XML dataset should act as nodes, under which relation two nodes should be linked to one another, and whether we display any other numerical or descriptive information over our graphâs edges and nodes by applying related visual effects. The following paragraphs outline entities, relationships and properties of the raw data, which we codify as Graphieâs data model in Python.\n\nPrimary data\n\nEach legislation page (either a whole item, or a part, or a section) on Ref. 2, is also offered as an XML file, which we refer to as the XML URL of that page. For instance, the full data of the Housing Act 2004 [18] is also available as an XML file [19] which can be referenced at footnote. Legislation XML files use the syntax of the Crown Legislation Markup Language (CLML) syntax and the associated schema. The XML files of the UK Legislation API are structured in two essential layers, the metadata layer and the content layer. Quoting from Ref. 20, âthe CLML model incorporates versioning and facilitates the notion of expressing changes over time which helps us to understand the underlying metadata semantics behind the surface contentâ. The metadata layer is formed by elements such as: title, publication, type, format and a rigid set of persistent URIs. The content layer contains a mix of hierarchical (Parts, Chapters) (see Figure 4) and textual tags (Pblock, P1group) (see Figure 5). In this paper, we only focus on the content layer. The complexity of the CLML schema is investigated in Ref. 21 and demonstrated in Figure 3 of Ref. 20.\n\nSubsequent P1group tags indicate the start of a section (see Figure 5).\n\nThe content of this section is formed in a sequence (section-specific) of embedded tags: P1group, P1, P1para, P2, P2para, P3, Text. Other sections, say section 193, due to their textual complexity, might have a different tag ordering, involving BlockAmendment tags.\n\nIn Graphie we wish to visualize Acts as networks, where each section is connected with other sections if and only if there is clear textual reference between them. Consider the following last line from section 194 â⊠so of this section so far as those subsections relate to section 138(2B) of the Housing Act 1985âŠâ, in Figure 12. In the same figure, the mentioned dependence is pictured as a link connecting the node representing the section 194 and the node representing the Housing Act 1985.\n\nIn the following section we define a minimal data model, sufficient for capturing both the hierarchical and textual elements of one Actâs XML structure.\n\nGraphie data model (or Graphie object)\n\nThe UK primary legislation is structured in Acts and each Act includes different levels of division (such as Parts, Chapters, cross-headings) in a certain order, a hierarchy outlined in Ref. 22. The Housing Act 2004 shown in Figure 6 includes seven numbered parts. Each Part contains numbered Chapters and cross-headings. Chapters are above cross-headings. Cross-headings are not numbered, are displayed in italics and highlight a group of sections beneath it. Part 1, as an example, contains Chapters and Chapters include cross-headings and each cross-heading contains sections. Part 3 instead, contains cross-headings but not Chapters.\n\nThe Act is organized in 7 Parts, following the Legislation Structure rules of Ref. 22. Sections in Part 1, shown on the left side, are located below cross-headings. Chapters in Part 1 include a series of cross-headings. Part 3 contains only cross-headings, but no Chapters.\n\nWithin the structural hierarchy defined in Ref. 22, sections are the lowest level of a piece of legislation. On the textual level, they contain sub-items organized in paragraphs that might include further numbered items. This textual structure is well captured by the related definitions from section 16 in Ref. 23. Section 194 (Figure 1) consists of: a number (194), a heading (âDisclosure of information as to ordersâŠâ) and its content. Section 194âs content is divided into four numbered subsections â (1), (2), etc. Each subsection starts with an introductory line and then contains two or more paragraphs. Subsection (3) contains three letter ordered paragraphs. Subparagraphs are usually numbered â(i), (ii)âŠâ and are nested within Paragraphs. For instance, consider section 97 [20] and its subsection (6) that contains two subparagraphs (i), (ii) within paragraph (b). Conjunctive words, such as âandâ or âorâ, might be used for transitioning between paragraphs and subparagraphs. The canonical fine substructure of a section is suggested in Ref. 23 such a refined definition is however missing from Ref. 22. In contrast, the UK Legislation XML documents, with few exceptions, are structured according to the section logic of Ref. 23.\n\nIn Figure 3, the legal hierarchy of Ref. 22 is included within the Data Model component and is represented by the following objects: Parts, Chapters and cross-headings, whereas the textual structure mentioned in Ref. 23 is embedded within the object Sections and includes a four-level object hierarchy: SubSections, Paragraphs, SubParagraphs and Lines. Lines are the lowest element of this textual hierarchy. Thus, instances of the Graphie model meet the three specific graph model criteria (hierarchy, sequence, reference) outlined in Ref. 11 (in section 2.1):\n\n1. Elements included in one instance of the Graphie model are hierarchically structured.\n\n2. One elementâs text value is always embedded within an object of a higher-level position. These objects should also be sequentially ordered.\n\n3. One elementâs text might contain words for expressing cross-references in other sections of the same Act (inbound references) or for pointing to sections from other Acts (outbound references).\n\nWe implement the details of the third criterion in Section 2.4. Methods refInSingleLine() and actsInSingleLine() are used for capturing a sectionâs cross-references and citations, respectively.\n\nIn this section, we will walk through the main methods of the Python XML parser given in Figure 3. The parser is developed using the beautifulsoup [21] library, a well known Python tool, which allows you to try out different web scrapping strategies.\n\nTable numbers refer to the obtained instances for each object after parsing Housing Actâs 2004 full XML content. The reported numbers are heuristically calculated and do not include textual information about sections declaring legislation amendments.\n\nConsider the âtable of contentsâ page (Figure 6) of the Housing Act 2004 [22]. As expected, both the underlying XML document and the displayed HTML page are structured in Parts, Chapters and cross-headings. Each sectionâs titles are hyperlinked, and each hyperlink points to one sectionâs whole web document. For instance, in Figure 6, the title of section 3, âLocal housing authorities to review housing conditions in their districtsâ, is located below the cross-heading âProcedure for assessing housing conditionsâ and points to the individual web page for section 3 [23].\n\nThe two fundamental components of our parser are the contents() method and the singleSection() method, both of which are defined below. Each method can be executed in isolation. The role of contents() is to map the legal hierarchy structure included in an Actâs âtable of contentsâ XML page into the following items of the Graphie Object: Parts, Chapters, cross-headings (mentioned in Section 2.1). Given a sectionâs XML URL and using the singleSection() method instead, we can capture and map the textual content of a section to the following elements of the Graphie Object: Sections, Paragraphs, SubParagraphs and Lines. Both methods are not Act-specific and will be discussed elsewhere in this article as well, such as in the context of Data Integrity (Section 2.3) and in the Transformation Service (Section 2.4) sections.\n\ncontents()\n\nThe âTable of Contentsâ page is where legislation readers could get an idea of how an Act is organized according to the legal hierarchy outlined in Ref. 22. The âTable of Contentsâ web page for the Housing Act 2004 (footnote 22) is divided into Parts, Chapters or cross-headings and sections. All sections have headings alongside their numbers, and are grouped within a Chapter or a cross-heading. The above structure is well identified in Figure 7, corresponding to an Actâs full data XML file: A Part is the root element for a group of Chapters. It has has a number and a title. Chapter tags have numbers and titles, too. A Chapter often includes cross-headings. A Pblock start tag indicates a cross-heading, and each cross-heading has a name. A cross-heading usually contains two or more sections. Sections are defined as P1group tags and always contain other elements such as Title, PNumber and DocumentURL elements.\n\nThe file contains information about same Actâs components: Parts, Chapters, cross-headings and sections.\n\nFor indexing an Actâs legal structure, we use the method contents(). The parsed table of contents of the Housing Act 2004 is shown in Figure 8. The method contents() is clearly able to handle the structural variety between Part 1 and Part 2 (no chapters, only cross-headings).\n\ncontents() is a function with one argument url. When contents() is called, we pass along the URL corresponding to one Actâs full data XML file. This URL is used inside the function for returning an instance of the Python object Parts that contains nested occurrences of the following objects: Chapters, cross-headings and Sections.\n\nAt this stage, one can easily declare a new array variable, say urls, for storing all the DocumentURL elements returned by contents() (see Figure 8). In the next section we discuss how we could fully XML-scrap an Actâs content by running the next defined method singleSection() for each DocumentURL in urls.\n\nsingleSection()\n\nThe aim of the singleSection() method is to map, applying the text-structural rules of Ref. 23, a sectionâs (or subsectionâs) content into an instance of the Python object Section defined in Section 2.1. In Figure 9, the object Section captures sectionâs 194 content in four instances of the object SubSections, reflecting the four numbered items in Figure 1. Each instance of SubSections consists of an array of Paragraphs objects, corresponding to the letter numbered lines. In our example, the SubSections object of the third item is composed of three Paragraphs, each paragraph corresponding to one item of the ordered list (a), (b) and (c).\n\nThe obtained Sections instance contains embedded objects (SubSections, Paragraphs, SubParagraphs and Lines) which are hierarchical and sequentially ordered: section 194 contains 4 subsections. Each subsection includes ordered paragraphs.\n\nFollowing legislation amendments, one sectionâs subsection can change another Actâs sections content.23 Consider that due to the use of the word âinsertâ in subsection (4) of section 185 [24] of the Housing Act 2004, section 155 of the House Acting Act 1985 is modified by inserting two new sections, sections 155A and section 155B. The word âsubstituteâ (see subsection (2) in section 185) is also used for such amendments. In our data model (see Figure 3) sections embed subsections and subsections incorporate paragraphs. Since amendments modify sections, they may be seen as superior objects to sections; on the other hand, their text is usually located within subsections. Our data model does not allow for sections to be subordinate to subsections, and is therefore unable to fully express amendments (see Section 2.3).\n\nsingleSection() is heuristic in nature and depends on the underlying structural variety of the Legislation XML schema. For achieving high levels of accuracy, singleSection()âs output should be manually or programmatically checked, as we explain in the next subsection.\n\nThe aim of the XML parser developed in Section 2.2 is at least twofold: firstly, we wish to speed up the data collection process and secondly, we plan to use the same parser for collecting data from other Acts. Data integrity is captured as a dedicated component in the flowchart of Figure 3, a component that requires the attention of data analysts well trained in various tools. In our scenario, prior to any visualization, we are looking to improve our XML parserâs output by incorporating related data quality observations.\n\nConsider a data quality engineer parsing the XML file of section 194 of the Housing Act 2004. Following the steps of our pipeline, they would need to inspect singlesection()âs output (displayed on a Jupyter window) against the original content of the same section on legislation.gov.uk (an HTML web page). Practically, this would require a visual comparison between two different pages on a browser. For facilitating speedy comparisons between parsed and original data, we developed a bespoke data integrity web tool. In Figure 10, the left frame displays one sectionâs parsed content. The same sectionâs original content (shown in Ref. 2) is located on the right frame. This blended visualization between original and parsed data makes their comparison easier and faster. Otherwise, we would have to keep two full screens open for comparing parsed and original data. Other key variables that we could use for evaluating our parserâs performance are the total number of subsections, paragraphs, sub paragraphs and single lines included within one section.\n\nOn the left, section 194âs parsed content. On the right, the same sectionâs content in Ref. 2.\n\nRegular expressions are useful for data engineering routines, also used in the preprocessing steps of Ref. 14. In Table 2 we report the frequency of a few important expressions after running Regex Search [25] on the Housing Act 2004âs one-page view (see footnote 18). Consider the regular expression âof the [aA-zZ]+ Actâ. This regex is used for finding all Act names mentioned within the Housing Act 2004 and reports 156 such instances (outbound references). In Table 5, prior to network visualization in Figure 12, we match these instances with the output obtained by our parserâs method: actsInSingleLine() (see Section 2.4). âsection [1-9]* or [1-9]â identifies those lines (say, subsection 1 in section 13 [26]) which include a section number (inbound reference), an âorâ and are followed by a numerical digit from 1 to 9, pointing to other sections. For the network shown in Figure 2, this is a useful pattern as it helps us to pin down and review edges corresponding to sections related by an âorâ conjunction.\n\nA data engineer can use these patterns as alternative metrics for reviewing our parserâs performance about cross-referencing and citations collection.\n\nAt this point of our data journey, we should expect that original XML legislation files are now accurately represented on memory as an instance of the Graphie Python object, defined in Section 2.1. In Figure 11, we show on the left the table of contents generated using a specific instance of the Graphie object, mirroring the Housing Actâs 2004 table of contents (footnote 22). The JSON file feeding the graph visualization of Figure 2 is also generated based on the aforementioned Graphie object.\n\nThe user visualizes section 7 after clicking on the lowest link within the table of contents of Part 1, located on the left panel (ToC). Subsection 2(d) (within the middle panel, (HC) contains a hyperlinked outbound reference to section 265 of the Housing Act 1985. This hyperlink enables users to display sectionâs 265 content on the right panel (ZIH). All of these panels - ToC, HC, and ZIH - are generated by the related methods shown in Table 3.\n\nThe transformation service is a library of Python methods for serializing the memory representation of an Act into customised JSON or HTML outputs. Checking data integrity over this marshalling process is a crucial part of a data engineerâs job. A data engineer will first automatically translate the on memory data into a specified format, and then they will consequently review the data validity of the obtained output. The relation between data integrity checks and the transformation service is also reflected in Figure 3.\n\nrefInSingleLine() and actsInSingleLine() are two fundamental functions of the transformation component of our pipeline. Each function identifies string cross-references (refInSingleLine()) or citations (actsInSingleLine()) to other Acts from a text string.\n\nLet us take as an example the single line of subsection 2(a) in section 7 (Figure 11): âsection 12 (power to serve an improvement notice)â. Use this line as an input parameter to refInSingleLine(), and section 12 is returned. Thus, section 7 and section 12 form an inbound reference that we also express as a dedicated link between node 7 (section 7) and node 12 (section 12) in Figure 2. The repeated application of method refInSingleLine() for each line of section 7 returns the following array: [âsection 12â:1, âsection 21â:1, âsection 29â:1]. The array indicates three single inbound references, from section 2 to sections 12, 21 and 29. Such calculated metadata values are also included in inbound.json [27], the underlying file of the inbound complexity network of Figure 2. Similarly, we have also found that the most frequently referenced section in Housing Act 2004, is section 102 (Table 4).\n\nIn Figure 2, on the left, the table of contents is generated by the method divNav(). The method htmlSingleSection() instead prints one single sectionâs content as a customized html paragraph of our platform. We use the methods refInSingleLine() and actsInSingleLine() for picking out which nodes (sections) should be linked together with an edge in Figures 2 and 12.\n\nA table showing the top 5 (out of 281) most referenced sections of the Housing Act 2004, their reported frequency after repeatedly applying refInSingleLine() to each single line of the same Act and the number of unique references after manually inspecting the Housing Act 2004âs full web content (see Section 2.3).\n\nSubsection 4(b)âs text in section 194 (Figure 1) is now passed as a parameter to actsInSingleLine() and âthe Housing and Regeneration Act 2008â title is returned. Thus, section 194 contains an outbound reference to the Act just mentioned. Call actsInSingleLine() on every line of section 194 and you will obtain the following array: [âHousing Act 1985â:4, âHousing and Regeneration Act 2008â:1], highlighting all the outbound references within section 194. The value 4 next to the key the Housing Act 1985 is a calculated metadata value that counts how many times the Housing Act 1985 is textually mentioned within section 194. The same value determines that edgeâs weight (henceforth, thickness) in Figure 12. One edgeâs thickness is also represented in outbound.json by the attribute âthickâ (same attribute is used in inbound.json). Thus, the outbound.json file populates information about nodes and links shown in Figure 12, a network that highlights the outbound connections between sections in Housing Act 2004 with sections from other Acts.\n\nIn this instance, the user could click on âthe Housing Act 1985â node and fetch on the right (ZIH panel), all the outbound references connecting the Housing Act 2004 with the aforementioned Act. Section numbers are hyperlinked and users can continue exploring sections from both Acts.\n\nUserMark [28] is a similar and well-matured mark-up tool, that allows users to create hypertext links from any text to AustLIIâs [29] legislation and High Court corpus.\n\n\n3. Use cases: Visualizations\n\nIn this section, we illustrate how Graphie could be used in practice. Graphie offers a few main functionalities: a One Page View (OPV) navigation, and two network visualizations. These representations could be a powerful tool for portraying the legal understanding of an ideal law practitioner that knows all the interconnections and shortcuts among different sections and Acts of the UKâs Statute Book. While similar visualizations could be obtained by using native Python or R network packages, like, ânetworkxâ [30], network analyses are usually published using browser friendly tools, such as D3.js, Cytoscape [31], KeyLines [32]. For example in Ref. 24, initial network analyses were completed in ânetworkxâ whereas their output was produced in Cytoscape. Other examples are networks in Sampo-UI, which exploit Cytoscape.js [33] mechanisms and features: zooming, panning, node sizing, node coloring, and directed edges. In Graphie we implemented, using native D3 libraries [34, 35] a novel network navigation framework that allows users to click on a node and display that nodeâs underlying textual content. Such advanced mouse functionalities are not supported by default in Cytoscape.js and require further development.\n\nThe landing page of Graphie, depicted in Figure 11 offers a One-Page View (OPV) of the Housing Act 2004. OPV displays a three-part canvas made up of the following panels: Table of Contents (ToC), highlighted contents (HC), and zoomed-in hyperlinks (ZIH). On the left, users can explore the ToC of the Housing Act 2004 by expanding the related tree hierarchy. By clicking on a ToC link, the user can see the actual content of the chosen section in the next panel (HC). Let us go over an example: A user clicks on the ToC link of section 7 (Figure 11) and that sectionâs content is shown within the HC area. Section 7 is connected with several other sections, and the user may now click on the link of section 265 of the Housing Act 1985 (outbound reference). This sectionâs content is then pulled up on the right ZIH panel. All mentioned panels (ToC, HC, ZIH) are visible at all times. Thus, OPV enables users to review different sections on one single page, without switching between different pages (as experienced in Refs. 2,3). Also, sections are not hyperlinked in one Actâs âfull textâ page on legislation.gov.uk (see footnote 18). Further, the OPV feature is not offered in Westlaw.3 In Graphie instead, section numbers are hyperlinked.\n\nFrom a technical point view, ToC displays the table of contents of the Housing Act 2004 as a nested ordered list. The HTML content of ToC is generated by the method divNav(), whereas sections in HC and ZIH are built by the method htmlSingleSection(). All mentioned methods are part of the Transformation service, see Section 2.4. For more details on the methods used for visualization, please refer to Table 3.\n\nBy clicking on the square navigation icon in Figure 2, a network graph representation of the Housing Act 2004 is opened. In this network, only sections of the same Act (inbound references) are connected and displayed. Each sectionâs raw XML data is analyzed for cross-references using the method refInSingleLine(), defined in Section 2.4. We use the former methodâs output for creating the json file inbound.json that includes clear information about this networkâs nodes and their connections. The same file is used for populating our network front-end library with data. The obtained network consists of 281 nodes and 517 edges. In inbound.json, each node record and each link record are associated with metadata variables, such as ânodeSizeâ and âthickâ. Edge thickness in Figure 2, depends on âthickâ values (also defined in Section 2.4). Node size instead, is set according to ânodeSizeâ and reflects the total count of references found within the Housing Act 2004 about that nodeâs section number. That is, section 194 appears only once in section 270, subsection 5(c). Thus, section 194âs node size is 1.\n\nNode coloring works as follows: each Part is assigned with a colour, and any nodes within this Part should be assigned the same color. For instance, section 1 of Part 1 is denoted as the blue node 1. All sections (nodes) of Part 1 are coloured in blue. The user can still click on one nodeâs circle, and display on the right (ZIH panel) their hyperlinked content.\n\nThe network reports strong connectivity between same coloured nodes, indicating that sections of the same Part are expected to be tightly linked together. Sections of Part 5 are content-less, thus excluded from this inbound complexity network.\n\nTable 5 lists the 5 most commonly mentioned Acts within the Housing Act 2004âs sections. There are 39 such Acts, obtained by applying the method actsInSingleLine() to each section of the Housing Act 2004. Each of these Acts is represented as a single, coloured node in Figure 12. This new network is obtained from the original inbound network (Figure 2) by also connecting sections of the Housing Act 2004 with the collected new (external) Acts. The resulting graph consists of 327 nodes and 671 edges and is obtained in Graphie by clicking on the star navigation icon. The user again can click on a node and display the underlying content of the chosen section.\n\nFor each Act, the column actsInSingleLine() displays their occurrences found by iteratively calling actsInSingleLine() (see Section 2.4) over the full content of the Housing Act 2004, whereas the next column âData Integrityâ shows the actual observed occurrences after our data integrity checks (say, regular expression searches elaborated in Section 2.3 and Table 2).\n\n\n4. Conclusions\n\nIn this paper, we designed and described a cross-Act pipeline for establishing a connection between the raw data provided by the UK Legislation API and our platformâs front-end network representations of Acts and Bills included in the UKâs Statute Book. Our networks reveal interesting associations between individual provisions, and also enable users to fully explore one Actâs content by just hovering over each node with the mouse. Nodes are associated with their underlying textual, hyperlinked content, and by clicking on one node the user can display this information on a dedicated panel alongside. Visiting different sections and hopping between provisions of an Act no long requires opening new pages via hyperlinks (or via copy-pasting of the new nodeâs address into the browser): the user can now remain on the same page and arrive at the information sought by simply clicking or hovering on their nodes of interest.\n\nIn our efforts to mold legislation from raw data into their final network visualizations, we faced the task of processing them carefully and accurately. We tried to automate this process as much as possible. Due to the structural variety of our underlying files and the need to calculate network-specific metadata instances (not included in our original data), we had to incorporate a supervised layer of data integrity and data quality checks.\n\nThe next challenge will be to apply the developed pipeline on a larger scale, and to go beyond the Housing Act we focused on for illustrative purposes. There are several ideas and further steps to be considered. First and foremost, we wish to use and test our parsing and data integrity routines against a larger volume and broader classes of Acts. The process of identifying references between sections (inbound or outbound) and populating related hyperlinks should be further improved and automated to reduce the need for human supervision. Due to the expected larger population of nodes, we might consider adjusting or creating new visualization tools to avoid cramming effects on the screen. Such refinements usually require long learning and development curves. Our experience working with the sections of the Housing Act 2004 has however convincingly demonstrated the proof of concept of a fully operational prototype for the network-based visualization of the UKâs primary legislation â beyond the classical text-only paradigm â which promises a fresh way to conceive, analyze, and represent legal texts in a user-friendly way.",
"appendix": "Data availability\n\nAll data used for this project were publicly available and downloaded from the UKâs legislation platform. 2 This data is available as both full XML files and in html formatted text, as discussed in more detail in the âPrimary Dataâ paragraph, in Section 2.\n\n\nReferences\n\nLoft P, Apostolova V: Acts and Statutory Instruments: the volume of UK legislation 1950 to 2016.2017. Reference Source\n\nNational Archives: Enacted UK Legislation.2022. Reference Source\n\nThomson Reuters: Westlaw UK â Online Legal Research.2022. Reference Source\n\nRuhl JB, Katz DM: Measuring, monitoring, and managing legal complexity. Iowa L. Rev. 2015; 101: 191.\n\nKoniaris M, Anagnostopoulos I, Vassiliou Y: Network analysis in the legal domain: a complex model for European Union legal sources. J. Complex Netw. 2017; 6: 243.\n\nVivo P, Katz DM, Ruhl JB, editors. The Physics of the Law: Legal Systems Through the Prism of Complexity Science. Front. Phys. 2021; 9. Publisher Full Text\n\nSmolyak A, Havlin S: Three Decades in Econophysics â from Microscopic Modelling to Macroscopic Complexity and Back. Entropy. 2022; 24: 271. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCodling E, Plank M, Benhamou S: Random walks in biology. J. R. Soc. Interface. 2008; 5: 813â834. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatz DM, Bommarito MJ: Measuring the complexity of the law: the United States code. Artif. Intell. Law. 2014; 22: 337â374. Publisher Full Text\n\nFörster Y-P, Annibale A, Gamberi L, et al.: Information retrieval and structural complexity of legal trees. J. Phys.: Complexity. 2022; 3: 035008. Publisher Full Text\n\nCoupette C, Beckedorf J, Hartung D, et al.: Measuring law over time: A network analytical framework with an application to statutes and regulations in the United States and Germany. Front. Phys. 2021; 9: 658463. Publisher Full Text\n\nErdelez S, OâHare S: Legal informatics: Application of information technology in law. Annu. Rev. Inf. Sci. Technol. 1997; 32: 367.\n\nHyvönen E, Tamper M, Ikkala E, et al.: Lawsampo portal and data service for publishing and using legislation and case law as linked open data on the semantic web.submitted.2021. Reference Source\n\nCoupette C, Hartung D, Beckedorf J, et al.: Law Smells: Defining and Detecting Problematic Patterns in Legal Drafting. Artif. Intell. Law. 2022. Publisher Full Text\n\nWilkinson M, Dumontier M, Aalbersberg I, et al.: The FAIR guiding principles for scientific data management and stewardship. Sci. Data. 2016; 3: 160018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHyvönen E: Digital Humanities on the Semantic Web: Sampo Model and Portal series. Semant. Web. 2022; 1â16. accepted. Publisher Full Text\n\nIkkala E, Hyvönen E, Rantala H, et al.: Sampo-UI, A Full Stack Javascript Framework for Developing Semantic Portal User Interfaces. Semantic Web - Interoperability, Usability, Applicability. Semant. Web. 2022; 13: 69â84. Publisher Full Text\n\nRobinson D, Yu H, Zeller W, et al.: Government data and the invisible hand. Yale J. L. Tech. 2009; 11: 159.\n\nOksanen A, Tuominen J, MÀkelÀ E, et al.: Semantic finlex: Transforming, publishing, and using Finnish legislation and case law as linked open data on the web.Peruginelli G, Faro S, editors. Knowledge of the Law in the Big Data Age, Front. Artif. Intell. Appl. 2019; 317: 212.\n\nKumar B, Mcgibbney L: A comparative study to determine a suitable representational data model for UK building regulations. J. Inf. Tech. Construction. 2013; 18: 20.\n\nKingâs Digital Lab: Taming the complexity of law: Modelling and visualization of dynamically interacting legal systems.2022. Reference Source\n\nNational Archives: FAQ, Enacted UK Legislation.2022. Reference Source\n\nGovernment of Western Australia, Department of the Attorney General, Parliamentary Counselâs Office: How to read legislation, a beginnerâs guide.2022. Reference Source\n\nFerolito B, do Valle IF , Gerlovin H, et al.: Visualizing novel connections and genetic similarities across diseases using a network-medicine based approach. Sci. Rep. 2022; 12: 14914. PubMed Abstract | Publisher Full Text | Free Full Text\n\nkclquantlaw: kclquantlaw/pipeline: Sofia, an offline, cross-Act, pipeline for parsing UKâs legislation XML documents (v1.0.0). [Code]. Zenodo. 2023. Publisher Full Text\n\nkclquantlaw: kclquantlaw/graphie: Graphie: A network-based visual interface for UKâs Primary Legislation (v1.0.0). [Code]. Zenodo. 2023. Publisher Full Text\n\n\nFootnotes\n\n1 Reported numbers till 2016, in Ref. 1.\n\n2 Available here: www.legislation.gov.uk/new\n\n3 https://www.nationalarchives.gov.uk\n\n4 The UK Legislation API, https://www.legislation.gov.uk/index\n\n5 https://www.legislation.gov.uk/developer/uris\n\n6 This means that section 194 includes multiple references to other statutes.\n\n7 https://graphie.quantlaw.co.uk/\n\n8 https://www.google.com/maps\n\n9 https://www.semanticscholar.org/\n\n10 https://pubmed.ncbi.nlm.nih.gov/\n\n11 https://arxiv.org/\n\n12 https://www.connectedpapers.com/\n\n13 https://d3js.org\n\n14 https://www.w3.org/TR/sparql11-query/\n\n15 https://js.cytoscape.org/ - a Graph theory library\n\n16 https://lettersampo.demo.seco.cs.aalto.fi/en/actors/faceted-search/network LetterSampo, a network of Historical Letters\n\n17 https://akatemiasampo.fi/en/people/faceted-search/network AcademySampo, a network of Finnish Academic People\n\n18 Available at https://www.legislation.gov.uk/ukpga/2004/34/data.xml\n\n19 https://www.legislation.gov.uk/ukpga/2004/34/data.xml, which we call the full data XML file.\n\n20 www.legislation.gov.uk/ukpga/2004/34/section/97\n\n21 https://www.crummy.com/software/BeautifulSoup/\n\n22 https://www.legislation.gov.uk/ukpga/2004/34/contents\n\n23 https://www.legislation.gov.uk/ukpga/2004/34/section/3\n\n24 www.legislation.gov.uk/ukpga/2004/34/section/185\n\n25 https://chrome.google.com/webstore/detail/chrome-regex-search/bpelaihoicobbkgmhcbikncnpacdbknn\n\n26 www.legislation.gov.uk/ukpga/2004/34/section/13\n\n27 graphie.quantlaw.co.uk/inbound.json\n\n28 http://www.austlii.edu.au/techlib/usermark/\n\n29 http://www.austlii.edu.au/\n\n30 https://networkx.github.io/\n\n31 https://cytoscape.org\n\n32 https://cambridge-intelligence.com/keylines/\n\n33 https://manual.cytoscape.org/en/stable/Navigation_and_Layout.html\n\n34 https://observablehq.com/@d3/force-directed-graph\n\n35 https://bl.ocks.org/mohdsanadzakirizvi/6fc325042ce110e1afc1a7124d087130"
}
|
[
{
"id": "172963",
"date": "25 May 2023",
"name": "Nicola Lettieri",
"expertise": [
"Reviewer Expertise Computational legal studies",
"law and complexity"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper presents Graphie, a tool implementing a network-based visual interface that allows exploring UK primary legislation in an intuitive and user-friendly way.\nAfter briefly introducing the rationale behind the research, the authors describe the system's building blocks, all gathered within an offline data pipeline that includes parsing, data modelling and visualisation functionalities.\nThe work is undoubtedly timely: the application of network analysis and visualisation techniques to legal corpora is a topic that, in recent years, has drawn the attention of different disciplinary fields (from law to computational legal studies) that still communicate relatively little.\n\nThe text is well-written, the structure is coherent, and the arguments are consistent. One relief can be made about the level of novelty of the proposal, a profile that is not sufficiently addressed in the related works section. Authors do not consider a series of papers that have already dealt with the application of network analysis and visualization techniques to complex legal corpora encompassing not only legislation and statutes but also legal literature, preliminary works and case law.\n\nFor the sake of completeness of inquiry, we draw authors' attention to several papers that have touched precisely on this type of application not only to enable more advanced and intuitive forms of access to legal information but also to enable a deeper understanding of the evolution of legal systems.\nThe authors could consider such a circumstance to better argue in favour of the originality of their work. Among the works closest to the topics the paper deals with:\nLettieri, N., Altamura, A., & Malandrino, D. (2017). The legal macroscope: Experimenting with visual legal analytics. Information Visualization, 16(4), 332-345. https://doi.org/10.1177/1473871616681374\n\nLa Cava, L., Simeri, A., & Tagarelli, A. (2022, July). LawNet-Viz: A Web-based System to Visually Explore Networks of Law Article References. In Proceedings of the 45th International ACM SIGIR Conference on Research and Development in Information Retrieval (pp. 3300-3305). - https://doi.org/10.1145/3477495.3531668\n\nLettieri, N., Altamura, A., Faggiano, A., & Malandrino, D. (2016). A computational approach for the experimental study of EU case law: analysis and implementation. Social Network Analysis and Mining, 6, 1-17. https://doi.org/10.1007/s13278-016-0365-6\n\nMoodley, K., Hernandez-Serrano, P. V., Zaveri, A. J., Schaper, M. G., Dumontier, M., & Van Dijck, G. (2020). The case for a linked data research engine for legal scholars. European Journal of Risk Regulation, 11(1), 70-93. - doi:10.1017/err.2019.51 .\n\nGórski, Å. (2021). Network Science in Law: A Framework for Polish Case-Law Citation Network Analysis. IT Professional, 23(5), 62-66.https://doi.org/10.1109/MITP.2020.3037354\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "175485",
"date": "30 May 2023",
"name": "Andreas Nishikawa-Pacher",
"expertise": [
"Reviewer Expertise Relevant for this paper: (a) general understanding of law and legal databases / legal informatics",
"(b) meta-science",
"including the data quality of metadata about scientific outputs and the role of APIs"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper presents an interesting tool that allows users to read legal texts such that cross-references (to other Acts, or to other sections of a given Act) are immediately clickable, with an optional visibility of such connections in network graphs.\nThe article does a great work in embedding the project into the wider field of legal informatics and of metadata-based network graphs. Similar efforts are referenced in a way that highlights the relevance of the current project. Furthermore, the method section is detailed and offers clear clues on how the project was created. While I personally lack some of the relevant methodical skills, the section sounds plausible, replicable and comprehensible even for a semi-layman like me.\nAs regards some critical words, I only have three minor issues to raise. They pertain more to the 'framing' of the text rather than the actual project per se, with the exception of the third remark on the UX of Graphie.\nFirst, section 3 outlines some use cases. Inter alia, \"[t]hese representations could be a powerful tool for portraying the legal understanding of an ideal law practitioner that knows all the interconnections and shortcuts among different sections and Acts of the UKâs Statute Book.\" -- I think this is an overstatement given how law (and lawyers) operate in terms, for instance, of implicit analogies. Relevant cross-references to other legal acts (or to other legally binding documents such as court decisions) are not always made explicit verbally in legal texts. They are thus difficult to put into metadata. One of the key tasks of a lawyer's education and work is, in fact, to flesh out such implicit cross-references in a creative way that can hardly be detected with (semi-)automated reference-extraction, at least according to my experience. I thus would not say that Graphie portrays the understanding of an ideal law practitioner, but only visualizes (and facilitates) the minimum baseline of tracing cross-references.\n\nIn other words, and to paraphrase another paper (p. 20 of an article titled 'Is Every Law for Everyone?'), an adequate visualisation of law's complexity cannot be \"achieved by just dumping [... legal] statutes into [Graphie]\"; or, putting legal texts into network graphs may be \"a necessary but by no means a sufficient condition for\" this purpose.\nSecond, a few optional ideas for the Discussion section. From the authors' viewpoint, what is the ideal future for reading/accessing/visualising primary legislation? Perhaps users having a huge touch-screen operating Graphie where they read legal documents side-by-side with network graphs which they can easily open cross-Act references with a single touch? Would that make one a \"better\" lawyer? (Sorry for the perhaps provocative question; I am just wondering about the final \"vision\" of this project.) Moreover, reading the final paragraph outlining the next challenges made me think about some concrete issues, e.g., automating the insertion of modifications of laws into Graphie; or: linking legal data across different countries and other polities; or: linking primary legislation with relevant court decisions are some of these possible/impossible to achieve, and what does this tell us about the limitations of this project? Finally, are there broader implications for legal informatics? E.g., did the authors experience some kind of missing metadata that they would wish to be implemented across every single country's official legal database, or are there even already elaborate and standardized metadata schemes that official legal databases could make use of so as to better allow for a scalability of Graphie?\nThe third minor aspect relates to some technicalities of Graphie itself (at least from the perspective of my own UI experience). For example, in network.html (the inbound network view), using my Chrome browser in 100% size leads the (supposedly?) \"right\" panel to be dropped into the bottom (or even outside) of the screen; it is only when I zoom out to 90% that I see the panel actually to the right. In addition, I find it slightly confusing that clicking on a reference leads me to the whole other section immediately, exiting the one I have been reading until then. For instance, 131(3) of the Housing Act tells me about a \"final management order under section 102(8)\", with some more text following after that reference, but if I seek to have a quick view of the referenced section 102(8), a click on it forces me to exit the paragraph that I have not even finished reading yet, leading me to section 102 without highlighting my relevant sub-part nr. 8 which is somewhere in the bottom of the browser, requiring me to scroll down a while, which I would only do if I actively read & memorized the correct paragraph number, which, in turn, I believe I should actually not do if the tool was to offer me an \"easier\" reading experience. In that way, the (assumed) task of smoothing the cross-reading of sections is not achieved in the optimal way, but the tool rather generates new cognitive burdens that one must take into account. While reading 131(3), perhaps the opening of a tiny box showing (a preview of?) 102(8) would be better. These are just very minor technical issues that in no way lessen the quality of the paper. The deeper implication is that a validation of the UI experience, or at least a discussion thereof, would be great.\nOverall, the article is excellent, the tool innovative & timely; my comments simply address three minor issues.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-236
|
https://f1000research.com/articles/12-232/v1
|
02 Mar 23
|
{
"type": "Research Article",
"title": "The present value of human life losses associated with COVID-19 and likely cost savings from vaccination in Kenya",
"authors": [
"Joses Kirigia",
"Germano Mwabu",
"Rose Nabi Deborah Karimi Muthuri",
"Germano Mwabu",
"Rose Nabi Deborah Karimi Muthuri"
],
"abstract": "Background: The study estimates the total present value (TPVKENYA ) of human lives lost due to COVID-19, total indirect costs attributed to COVID-19 mortality, total direct costs of all COVID-19 cases, and projected cost savings due to COVID-19 vaccination as of 25 July 2022. Methods: We used a human capital approach (HKA) model to estimate TPVKENYA . The indirect cost of COVID-19 (ICi=1,..,6)  for each of the six productive age groups equals the present value multiplied by the relevant employment-to-population ratio. The direct cost (DCi=1,..,4)\nfor each of the four disease severity categories (asymptomatic, mild/moderate, severe, critical) is the product of the number of COVID-19 cases in a severity category and the average total direct cost per patient. The total direct cost saving equals the number of infections averted with vaccination multiplied by the average total direct cost per patient treated. The total indirect cost saving equals the number of COVID-19 deaths prevented with vaccination multiplied by the average total indirect cost per death. Results: The cumulative 5670 human life losses had a TPVKENYA  of Int$268,408,687 and an average total present value of Int$47,338 per human life. A re-run of the HKA model with (a) discount rates of 5% and 10% reduced TPVKENYA by 16% and 39%, respectively; (b) Africa's highest life expectancy of 78.76 years and world's highest life expectancy of 88.17 years increased TPVKENYA by 79% and 129%, respectively; (c) excess mortality of 180,215 increased TPVKENYA by 3,078%. Total indirect and direct costs of COVID-19 were Int$36,833 per death and Int$1,648.2 per patient/case, respectively. The 30% target population's COVID-19 vaccination coverage may have saved Kenya a total cost of Int$ 1,400,945,809. Conclusions: The pandemic continues to erode Kenya's human health and economic development. However, scaling up COVID-19 vaccination coverage would save Kenya substantial direct and indirect costs.",
"keywords": [
"COVID-19",
"value of life",
"direct cost",
"indirect cost",
"cost savings from vaccination"
],
"content": "1. Background\n\nKenya is on the Eastern side of the African continent. It is one of the East African Community's seven member states (including the Democratic Republic of the Congo, Burundi, Rwanda, South Sudan, Uganda, and the United Republic of Tanzania).1 In 2022, it had an estimated population of 56,206,851 people,2 a total gross domestic product (GDP) of International Dollars (Int$) 293.423 billion, and a GDP per capita of Int$ 5762.003.3 In 2021, the country had a Gini Coefficient of 40.8.4 The national income shares held by the poorest 40 per cent, richest 10 per cent, and richest one per cent were 16.5%, 31.6%, and 15.2%, respectively.\n\nAccording to the World Bank, during the global coronavirus disease (COVID-19) pandemic, the real GDP contracted by 0.4% in 2020 compared with 5.4% in 2019.5 The first case of COVID-19 was confirmed in Kenya on 12 March 2020.6 As of 25 July 2022, Kenya had reported a cumulative total of 337,339 coronavirus disease (COVID-19) cases, consisting of 330,910 recoveries, 5,670 deaths and 759 active cases.7 However, the level of testing in the country has been low. For example, by 25 July 2022, Kenya had conducted 67,769 COVID-19 laboratory tests per million population compared with 426,031 and 7,614,872 per million population in South Africa and the United Kingdom (UK), respectively.8 Therefore, there is a likelihood that the COVID-19 burden in Kenya is substantively underreported.\n\nThe morbidity and mortality from COVID-19 in Kenya could be attributed to underperformance in four health-related systems. First, the sub-optimal national health system (NHS). For instance, in 2019, Kenyaâs average universal health coverage (UHC) service index was 56 on a scale of 0 to 100 (target).9 It signifies an overall gap in essential health services coverage of 44, which is attributed to deficits in its constituent components of 65 in the UHC coverage sub-index (UHCCSI) on service capacity and access, noncommunicable diseases (NCDs) UHCCSI of 28, infectious diseases (IDs) UHCCSI of 47, and reproductive, maternal, neonatal and child health UHCCSI of 27.\n\nSecond, weaknesses in Kenyaâs integrated disease surveillance system (IDSS) as reflected in gaps in the implementation of International Health Regulations (IHR) capacities.10 For example, as shown in Table 1, in 2020, Kenyaâs average 13 IHR core capacity score was 44 on a scale of 0 to 100, denoting an implementation gap of 56%.11\n\nNone of the 13 IHR capacities listed in Table 1 had an optimal score of 100. The IHR capacities of human resources and radiation emergencies had gaps of 80%; legislation and financing, health service provision, points of entry, chemical events, and coordination/national focal point functions had gaps of 60%; the national health emergency framework had a gap of 53%; surveillance had a gap of 50%; laboratory, risk communication, food safety, zoonotic events and the human-animal interface had a gap of 40%.\n\nThe third is the underperformance of systems tackling social determinants of health (SDH), such as education, food, shelter, sanitation and water. For example, in 2018, the literacy rate was 81.54% among people aged 15 years and above, meaning about 5,746,249 people were illiterate.12\n\nIn 2022, according to Concern Worldwide and Welthungerhilfe,13 Kenya had a Global Hunger Index (on a scale of 0 denoting no hunger and 100 being the worst) score of 23.5, which signified a severe level of hunger. In addition, about 32.2% of the population is undernourished, the prevalence of wasting in children under five years is 4.8%, and the prevalence of stunting in children under five years is 23.6%.\n\nConcerning shelter, 46.1% of the urban population lived in slum households in 2018, characterised by a lack of access to improved sanitation and water, plus a lack of sufficient living area and quality/durability of structure.14 According to the World Health Organization (WHO), in 2020, 19.5% of the population primarily relied on clean fuels and technologies for cooking.15\n\nIn 2020, 26.76% of the total population had basic handwashing facilities at home, 61.63% used basic drinking water services, and 32.7% used basic sanitation services.16\n\nFourth, in 2019, Kenya had a national health research system (NHRS) barometer score of 85%,17 denoting the existence of a performance deficit of 15%. An optimally performing NHRS timeously generates pertinent evidence and facilitates its use in policy, planning, innovation, and development of products to combat pandemics.18\n\nThe sub-optimal performances of the NHS, IDSS, SDH, and NHRS may be attributed to both underinvestment and inefficient allocation and use of systems resources. For example, in 2019, Kenyaâs current health expenditure per capita of US$8319 was 43% below the target recommended for lower-middle-income countries by Stenberg et al.20 of US$146 per person to attain the health-related Sustainable Development Goal 3.21\n\nMoreover, the Kenya Health Policy 2014â203022 and the Health Sector Strategic and Investment Plan23 underscore the need to increase the cost-effectiveness and cost-efficiency of resource allocation and use.22 It calls for concerted action by the Ministry of Health to mount evidence-based advocacy within the government (in the context of the âHealth-in-all-Policiesâ approach), the Ministry of Finance, the Ministry of Labour, and other relevant ministries), the domestic private sector, and stakeholders to augment investments to bridge health-related systemic gaps.24\n\nAccording to Card and Mooney,25 explicit monetary valuation of human life is a vital component of a decision theory model for allocating scarce health development resources rationally. Rice26 explains that its essential to translate the magnitude of disease in dollar terms because it is the universal language of decision-makers in the policy arena. Some studies have applied the human capital approach to monetarily value human life losses associated with COVID-19 in Brazil,27 Canada,28 China,29 France,30 Germany,31 India,32 Iran,33 Italy,34 Japan,35 Mauritius,36 South Africa,37 Spain,38 Turkey,39 UK,40 and United States of America (USA).41 There is a dearth of such economic evidence for Kenya, yet it is still needed for advocacy. In addition, although Barasa et al.42 assessed the unit costs for COVID-19 case management in Kenya, no study has estimated the potential total cost savings due to COVID-19 vaccination. The study reported in this paper was a modest attempt to bridge those knowledge gaps.\n\nThe specific study objectives were to estimate the following:\n\na) The total present (discounted) value of reported human lives lost in Kenya due to COVID-19, as of 25 July 2022.\n\nb) The total indirect costs (productivity losses) attributed to reported mortality from COVID-19, as of 25 July 2022.\n\nc) The total direct costs (health system inputs costs) incurred in caring for all the COVID-19 cases reported, as of 25 July 2022.\n\nd) The potential/projected direct and indirect cost savings due to COVID-19 vaccination, as of 25 July 2022.\n\n\n2. Methods\n\nThe valuation of human life cross-sectional study on Kenya was for the 5,670 deaths the government reported between 12 March 2020 and 25 July 2022.7 The 47 Kenya's administrative counties' share of COVID-19 deaths were as follows:\n\n⢠Fewer than two deaths in Baringo, Elgeyo Marakwet, Homa Bay, Kirinyaga, Nyamira, Nyandarua, Samburu, Tana River, Tharaka Nithi, and West Pokot.\n\n⢠Two to 10 deaths in Bomet, Bungoma, Embu, Isiolo, Kakamega, Lamu, Kisii, Kitui, Mandera, Marsabit, Nandi, Trans Nzoia, Vihiga, and Wajir.\n\n⢠Ten to 20 deaths in Garissa, Laikipia, Meru, Muranga, Kerichu, Kwale, Siaya, Taita Taveta, and Turkana.\n\n⢠Twenty-one deaths and above in Busia, Kiambu, Kajiado, Kilifi, Kisumu, Machakos, Makueni, Migori, Mombasa, Nairobi city, Nakuru, Narok, Nyeri, and Uasin Gishu.\n\nThe indirect costs calculation was for the 5,586 reported deaths in the economically productive age bracket of 15 years and above.7,8 Also, the direct cost estimation was for a cumulative total of 337,339 COVID-19 cases reported, as of 25 July 2022.7,8\n\nThe direct cost savings estimations encompassed the projected 182,423 COVID-19 infections averted, assuming 30% coverage of the target population (15 years and above) of 31,786,253 with the Oxford-AstraZeneca vaccine.52,53 The indirect cost savings calculations included the projected 29,872.27 deaths prevented, assuming 30% coverage of the target population with the COVID-19 vaccine.52â54\n\n2.2.1 Model for estimating the present value of reported human lives lost\n\nAccording to Culyer,43 there are three main approaches for valuing human life: the human capital approach (HKA), the social decisions approach or implied values approach (IVA), and the contingent valuation approach (CVA) or willingness-to-pay approach. First, the HKA assesses the value of a human life lost from any cause (disease or injury) in terms of the discounted expected money worth of goods and services lost by society due to their premature death. Weisbrod44 defines the present value of a human being as their discounted expected future income stream net of their consumption.\n\nSecond, the IVA (or revealed preference approach as observed in actual choices) infers values from actual past life-saving choices (or decisions) in the public sector.43\n\nThird, the CVA seeks to establish through a questionnaire survey the maximum amount of money individuals are willing to pay for small reductions in the risk of death they face concerning any cause, e.g., COVID-19.43 Unfortunately, according to Robinson et al.,45 there are few or no direct estimates of value per statistical life for most low- and middle-income countries which employ the willingness-to-pay (WTP) approach to assess the willingness of those affected by public health challenges (such as COVID-19) to trade their income for small reductions in risk of death.\n\nDue to the availability of data on GDP per capita and current health expenditure per person, we applied HKA to estimate the total present value (TPVKENYA) of human lives lost in Kenya due to COVID-19 as of 25 July 2022. A similar approach has been used in Brazil,27 Canada,28 China,29 France,30 Germany,31 India,32 Iran,33 Italy,34 Japan,35 Mauritius,36 South Africa,37 Spain,38 Turkey,37 UK,40 and USA.41\n\nThe TPVKENYA is a summation of the present value of human lives lost in age groups PVi=1,..,7 0â9 years, 10â19 years, 20â29 years, 30â39 years, 40â49 years, 50â59 years, and 60 years and above. Formally27â41:\n\nThe PVi=1,âŠ,7 for each of the seven age groups was a sum of the product of undiscounted years of life lost (UYLL), net GDP per capita, and COVID-19 deaths in a specific age group.27â41 Formally:\n\nWhere: ât=1T is the summation from the undiscounted year one of life lost [UYLL] (t = 1) to the final UYLL (T) in a specific age group, where the age groupâs total number of UYLL equals average life expectancy at birth for Kenya ALEKENYA minus average age at onset of death AADi=1,âŠ,7; r is the discount rate of 3%; 11+rt is the discount factor formula; COVIDDKENYA is the total number of COVID-19 deaths in Kenya between 12 March 2020 and 25 July 2022; PDi is the ith age group share of COVID-19 deaths.\n\nIn Equation 2, all the years lost due to COVID-19, even those below the minimum working age of 15 years, are valued. We did this because Subsection 2.2.1 primarily concerns the monetary valuation of human lives lost at all ages, irrespective of productivity.\n\n2.2.2 Model for estimating productivity losses (indirect costs) attributed to reported mortality from COVID-19\n\nKenyaâs total indirect cost or productivity loss TICKENYA is a summation of indirect costs in economically productive age groups ICi=1,..,6, i.e. 1 = 15â19 years, 2 = 20â29 years, 3 = 30â39 years, 4 = 40â49 years, 5 = 50â59 years, and 6 = 60 years and above.\n\nFormally27â41:\n\nThe ICi=1,..,6 for each age group, 1, 2, 3, 4, 5 and 6 equals the present value PVi=1,..,6 multiplied by the relevant employment-to-population ratio EPRi=2,..,6 and ith age group share SHAREi=1. Formally:\n\nThe PVi=1,..,6 was computed as explained in Subsection 2.2.1. The EPRi=1,..,6, the proportion of a countryâs working age population employed, was obtained from the Kenya National Bureau of Statistics (KNBS) quarterly labour force report.46 SHAREi=1,âŠ,6 is the proportion of the indirect cost to be apportioned to the age group, which varies from 0 to 1.\n\nAccording to the International Labour Organization (ILO) Minimum Age Convention No. 138, the minimum working age â⊠shall not be less than the age of completion of compulsory schooling and, in any case, shall not be less than 15 years (Article 2)â.47 Therefore, since the minimum working age in Kenya is 15 years, we assume that 50% of deaths from COVID-19 in age group 1 (10â19 years) were between 15 and 19 years. Therefore, the IC1 for age group 1 (15â19 years) equals the present value PV1 multiplied by the group employment-to-population ratio EPR1 multiplied by 0.5, i.e., age groupâs share SHAREi=1.\n\n2.2.3 Model for estimating the total direct cost of reported COVID-19 cases care\n\nThe total direct costs of COVID-19 (TDCKENYA) encompass the value of quantities of inputs used by the NHS to provide appropriate health interventions to different disease severity categorises. For instance, TDCKENYA is the sum of direct costs across the four disease severity categories DCs=1,..,4, i.e. 1 = home-based isolation and care for asymptomatic cases, 2 = hospital/isolation centre care for mild/moderate cases, 3 = hospital high dependency unit care for severe cases, and 4 = hospital intensive unit care for critical cases.\n\nFormally:\n\nThe DCi=1,..,4 for each disease severity category 1, 2, 3, and 4 is the product of the number of COVID-19 cases in a severity category CASESs=1,..,4, average total direct cost per patient ADCs=1,..,4, and conversion rate from US$ to Int$ CR. Formally:\n\nThe ADCs=1,..,4 estimates from Baraza et al.42 were used to estimate the cost of managing the four clinical categories of COVID-19 cases (see Table 2). The health system input costs by Baraza et al.42 included human resources for health, health worker transport, accommodation and overheads, pharmaceuticals (e.g. medicines), non-pharmaceuticals (fluids, oxygen, devices), COVID-19 tests, other laboratory tests, radiology, personal protective equipment, oxygen therapy, and capital items (e.g. buildings, medical equipment and vehicles).\n\n* Number of cases from Worldometers2 and Republic of Kenya.7\n\n** Total cost per patient from Barasa et al.42\n\n*** Kenyaâs GDP in 2022 is US$116.641 billion, which is equivalent to Int$293.423 billion from International Monetary Fund (IMF).3 Thus, the CR from US$ to Int$ equals 2.51560771941256, i.e. Int$293.423 billion divided by US$116.641 billion.\n\n2.2.4 Model for estimating potential direct and indirect cost savings due to COVID-19 vaccination\n\nThe potential savings associated with vaccination equals total direct cost savings TDCSKENYA plus indirect cost savings TICSKENYA.\n\n2.2.4.1 Direct cost savings model\n\nThe TDCSKENYA equals the number of COVID-19 cases averted with vaccination AVERTEDINFECTIONS multiplied by the average total direct cost per patient treated ATDC. In other words:\n\n2.2.4.2 Indirect cost savings model\n\nThe TICSKENYA equals the number of COVID-19 deaths prevented with vaccination COVID19DPREVENTED multiplied by the average total indirect cost per death ATIC. Formally:\n\nTable 3 shows the data and sources used in the Kenya analysis.\n\n2.4.1 The total present value of reported human lives lost in Kenya due to COVID-19, as of 25 July 2022\n\nExcel Software (Microsoft, New York) was employed to estimate Equations 1 and 2. The process involved seven steps.\n\nStep 1: Computation of the undiscounted years of life lost\n\nAs depicted in Table 4, the UYLL for each of the seven age groups (1 = 0â9 years, 2 = 10â19 years, 3 = 20â29 years, 4 = 30â39 years, 5 = 40â49 years, 6 = 50â59 years, 7 = 60 years and above) were computed through subtraction of AADk per age group from Kenyaâs ALEKENYA.\n\nStep 2: Computation of the DYLL\n\nApproximation of the DYLL at a 3% rate for each age group as a product of UYLL and the appropriate discount factor.27â41 For instance:\n\n⢠First DYLL in age group 20â29 = Discount factor à UYLL = [1/(1 + 0.03)1] = 0.970873786 à 1 = 0.970873786;\n\n⢠Thirtieth DYLL in age group 20â29 = Discount factor à UYLL = [1/(1 + 0.03)30] = 0.41198676 à 1 = 0.41198676;\n\n⢠Forty-third DYLL in age group 20â29 = Discount factor à UYLL = [1/(1 + 0.03)43] = 0.280542936 à 1 = 0.280542936.\n\n⢠Summation of the DYLL from year 1 to 43 yields 23.98190213 DYLL per human life lost in the age group 20â29.\n\nThe total number of DYLL in the age group 20â29 equals DYLL per human life lost (23.98190213) multiplied by the number of deaths (150) in the age group, i.e. 23.98190213 à 150 = 3,597.3. Table 5 depicts the DYLL per age group due to COVID-19 in Kenya at 3%, 5%, and 10% discount rates.\n\nStep 3: Assessment of Kenyaâs net GDP per person in 2022 International Dollars F2\n\nThe net GDP per person NGDPPPKENYA equals GDP per capita GDPPCKENYAminus current health expenditure per capita CHEPCKENYA.27â41 The 2022 GDPPCKENYA was Int$ 5762.003. Kenyaâs most updated data on CHEPCKENYA were for 2019.19 The CHEPCKENYA for 2022 was forecasted utilising values of Int$185.41142273 in 2018 and Int$207.61849976 in 2019.19 Applying the annual growth rate of 11.9771892707702%, the forecast for the 2020 CHEPCKENYA equals Int$ 232.485360437389; forecast for the 2021 CHEPCKENYA equals Int$ 260.330572083807; and forecast for the 2022 CHEPCKENYA equals Int$ 291.510857431964. Thus, the NGDPPPKENYA = GDPPCKENYA â CHEPCKENYA = Int$5762.003 â Int$291.510857431964 = Int$5,470.49.\n\nStep 4: Distributing the COVID-19 deaths across seven age groups\n\nThis was accomplished through multiplication of the 5670 reported cumulative COVID-19 deaths as of 25 July 2022 in Kenya COVIDDKENYA by the respective age groupâs proportion (PDk).7 Therefore, the number of deaths accrued per age COVIDDk=1,âŠ,7 was:\n\n(a). COVIDD0â9 = COVIDDKENYAÃPD0â9=5670Ã0.010934744=62;\n\n(b). 10â19 years = COVIDDKENYAÃPD10â19=5670Ã0.007760141=44;\n\n(c). 20â29 years = COVIDDKENYAÃPD20â29=5670Ã0.026455026=150;\n\n(c). 30â39 years = COVIDDKENYAÃPD30â39=5670Ã0.072486772=411;\n\n(d). 40â49 years = COVIDDKENYAÃPD40â49=5670Ã0.114991182=652;\n\n(d). 50â59 years = COVIDDKENYAÃPD50â59=5670Ã0.181834215=1031;\n\n(e). 60 years and above = COVIDDKENYAÃPD60and above=5670Ã0.585537919=3320.\n\nStep 5: Computation of total present value of human lives lost per age group (PVk)\n\nThe PVk=1,âŠ,7 was computed through the multiplication of DYLL per person in an age group, NGDPPPKENYA, and the number of deaths in an age group COVIDDk=1,âŠ,7.27â41 For instance, the PV20â29 for age group 20â29 years was obtained from the multiplication of DYLL per person in the age group of 23.982, NGDPPPKENYA of Int$5,470.49, and COVIDD20â29 of 150. Therefore, PV20â29=23.982Ã5470.49Ã150=Int$19,678,994.\n\nStep 6: Distribution of Kenya's total present value by administrative counties\n\nThe TPVKENYA was shared across 47 administrative counties (TPVj=1,âŠ,47) through the multiplication of TPVKENYA by each countyâs proportion of COVID-19 deaths.27â41 For example, given TPVKENYA is Int$ 268,408,687 and PDNAIROBI is 0.588382574 (Table 3), the share of TPVKENYA for Nairobi County equals Int$157,926,994.\n\nStep 7: Sensitivity analysis\n\nOne-way sensitivity analysis was conducted through re-estimation of the economic model five times, assuming (i) a 5% discount rate, (ii) a 10% discount rate, (iii) Africaâs highest average life expectancy at birth of 78.76 years (Algeria females),48 (iv) the world highest life expectancy of 88.17 years (Hong Kong females),48 and (v) projected excess COVID-19 mortality of 180,217.4721 deaths as of 25 July 2022 in Kenya (COVIDDKENYA).7,8,50 How was the latter forecasted?\n\nThe COVID-19 Excess Mortality Collaborators49 estimated that the actual number of COVID-19-associated deaths may have been far more significant than those reported due to Kenya's weak death registration system. According to COVID-19 Excess Mortality Collaborators,36 by 31 December 2021, the reported COVID-19 deaths in Kenya were 5380 (5.7 per 100,000), and the estimated excess deaths were 171,000 (181.2 per 100,000). The ratio between excess mortality and the reported COVID-19 mortality rate was 31.78438662. The projected excess number of COVID-19 deaths as of 25 July 2022 was 180,217.4721, i.e. 5670 reported deaths as of 25 July 2022 multiplied by 31.784.\n\n2.4.2 The indirect costs (productivity losses) attributed to reported mortality from COVID-19\n\nEquations 3 and 4 were built into an Excel spreadsheet and used to estimate the indirect costs following the seven steps below.\n\nStep 1: Search the ILO website for the minimum working age in Kenya.47\n\nStep 2: Delineate the economically productive age groups46: 1 = 15â19 years, 2 = 20â29 years, 3 = 30â39 years, 4 = 40â49 years, 5 = 50â59 years, and 6 = 60 years and above.\n\nStep 3: Extract the present values for each of the six economically productive age groups from the results obtained following procedures explained in Subsection 2.2.1.\n\nStep 4: Extract the employment-to-population ratio for each productive age group EPRi=1,..,6 from KNBS quarterly labour force report of 2021.46\n\nStep 5: Ascertain the proportion (ranging from 0 to 1) of the indirect costs to be apportioned to the age group. Age groups coded 2 to 6 were allotted a share of 1 because all persons in those age groups were presumed to be potentially economically productive. Given that in the age group 10â19, only persons aged 15 to 19 years (i.e., five years) are potentially productive, we divided five years by 10 years (number of years in the age group) and obtained a value of 0.5, as the age group SHAREi=1.\n\nStep 6: Estimate the ICi=1,..,6 for each age group 1, 2, 3, 4, 5 and 6 by multiplying the present value PVi=1,..,6, the relevant employment-to-population ratio EPRi=1,..,6, and the share of the indirect cost to be apportioned to the age group SHAREi=1,..,6.\n\nStep 7: Sum up the six age groups' indirect costs to derive Kenya's total indirect cost or productivity loss TICKENYA.\n\n2.4.3 The direct cost attributed to reported COVID-19 cases\n\nEquations 5 and 6 were built into an Excel spreadsheet and used to estimate the direct costs following the six steps below.\n\nStep 1: Determine the share of the total COVID-19 cases reported in Kenya by four disease categories: 1 = asymptomatic cases on home-based care, 2 = mild/moderate cases on hospital/isolation centre care, 3 = severe cases on hospital high dependency unit care, and 4 = critical cases on hospital intensive unit care. According to the Kenya Ministry of Health COVID-19 daily report 940,51 out of the total number of COVID cases, 73.06% were from home-based care, and 26.94% were from various health facilities. Out of the total COVID-19 cases treated at health facilities, 72.9% were mild-to-moderate cases admitted in a general ward, 12.1% were severe cases treated in a high dependency unit, and 15.0% were treated in an intensive care unit.\n\nStep 2: Estimate the number of COVID-19 cases per disease category CASESs=1,..,4 by multiplying the total number of reported cases (330,910) by the share for each clinical category (obtained from Step 1). For instance, category 1 = 330,910 cases à 0.7306 = 241,762.8; category 2 = 330,910 cases à 0.2694 à 0.729 = 64,988.3; category 3 = 330,910 cases à 0.2694 à 0.121 = 10,786.8; category 4 = 330,910 cases à 0.2694 à 0.15 =13,372.1.\n\nStep 3: Search in âPubmed.comâ for a published Kenyan study documenting the average total direct cost per patient per clinical category ADCs=1,..,4. The search revealed a study by Barasa et al.42 that reported ADCs=1,..,4 (see Table 2).\n\nStep 4: Derive a rate for converting CR unit costs expressed in US Dollars (US$) into International Dollars (Int$) using GDP data from the IMF World Economic Outlook Database.3 As shown in Table 2 in 2022, Kenyaâs GDP in 2022 was US$116.641 billion, equivalent to Int$293.423 billion.3 Thus, the CR from US$ to Int$ equals 2.51560771941256, i.e. Int$293.423 billion divided by US$116.641 billion.\n\nStep 5: Estimate the direct cost per disease severity category DCi=1,..,4 by multiplying the number of COVID-19 cases in a severity category CASESs=1,..,4 from Step 2 by the respective average total direct cost per patient ADCs=1,..,4 from Step 3 and CR from Step 4.\n\nStep 6: Calculate Kenyaâs total direct cost TDCKENYA through summation of direct cost (obtained in Step 5) across the four disease severity categories DCs=1,..,4.\n\nOur study estimates the potential savings from COVID-19 vaccination using actual population coverage of 30%, as of 25 July 2022, and the potential direct and indirect cost savings of the projected COVID-19 cases and deaths averted due to vaccination.\n\nAs explained by the COVID-19 Excess Mortality Collaborators49 (Subsection 2.4.1), the actual number of COVID-19-associated deaths may have been underestimated by a ratio of 31.784. For this reason, we decided to base the estimation of potential direct and indirect cost savings from COVID-19 vaccination on the projected total number of cases and deaths as of 25 July 2022.\n\n2.5.1 Expected savings in total direct costs due to COVID-19 vaccinations\n\nEquations 7, 8, 9 and 10 were built into an Excel spreadsheet and used to estimate the expected total direct cost savings attributable to vaccination with the Oxford-AstraZeneca vaccine following seven steps.\n\nStep 1: Obtain target population (15 years and above) of 31,786,253 for Kenya from the Kenya COVID-19 vaccination programme daily situation report dated 26 July 2022.52\n\nStep 2: Search PubMed for an epidemiological study on COVID-19 vaccine efficacy. The research revealed a study by Voysey et al.53 that found \"Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4â74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group (p. 881)\". Their study was a pooled analysis of four randomised trials (Brazil, South Africa, and the UK) with 8597 participants receiving the Oxford-AstraZeneca vaccine and 8581 receiving the control vaccine or saline.\n\nStep 3: Use the evidence in Step 2 to estimate the COVID-19 infection risk withoutIRControl and with Oxford-AstraZenecaIRAZ in the Voysey et al.53 pooled analysis of randomised trials in Brazil, South Africa, and the UK (Table 6). Infection risk in a group equals the number of infected persons divided by group size.\n\nStep 4: Estimate the number of people in Kenya expected to contract COVID-19 without vaccinationPoPIwithout. The PoPIwithout was obtained by multiplying the target population TPoPKENYA of 31,786,253 by the IRControl of 0.02890106.52,53 Thus, PoPIwithout equals 918,656.405, i.e. TPoPKENYAÃIRControl=31,786,253Ã0.02890106.\n\nStep 5: Approximate the number of people in Kenya expected to contract COVID-19 even after being fully vaccinated with the Oxford-AstraZeneca vaccine PoPIwith. The PoPIwith was derived by multiplying respective TPoPKENYA by IRAZ of 0.00977085.52,53 Therefore, PoPIwith equals 310,578.71, i.e. TPoPKENYAÃIRAS=31,786,253Ã0.00977085.\n\nStep 6: The number of infections averted, assuming 30% population coverage, equals the difference between PoPIwithout and PoPIwith, multiplied by 0.30 (30% coverage).52 Since PoPIwithout = 918,656.41 (from Step 4) and PoPIwith= 310,578.71 (from Step 5), infections averted through 30% vaccination coverage with Oxford-AstraZeneca equals 182,423.31, i.e. (918,656.41 â 310,578.71) à 0.30.\n\nStep 7: The total direct cost savings expected from vaccination equals the number of infections averted (from Step 6) multiplied by the average total direct cost per patient. For instance, the expected savings in Kenya equals Int$300,668,273, i.e. 182,423.31 infections averted (from Step 6) multiplied by the average total direct cost per patient of Int$1,648.19.\n\n2.5.2 Expected savings in projected total indirect costs due to COVID-19 vaccination\n\nEquations 11, 12, 13 and 14 were built into an Excel spreadsheet and used to estimate the expected savings in total indirect costs due to COVID-19 vaccination following the six steps below.\n\nStep 1: A search in the PubMed.com database for COVID-19 vaccine effectiveness in reducing the risk of death revealed an article by Bernal et al.,54 which attempted âto estimate the real-world effectiveness of the Pfizer-BioNTech BNT162b2 and Oxford-AstraZeneca ChAdOx1-S vaccines against confirmed covid-19 symptoms, admissions to hospital, and deathsâ (p.1).\n\nStep 2: Utilise the evidence in Step 1 to calculate the risk of COVID-19 resulting in death among the unvaccinatedDRunvaccinated and those vaccinated with the Pfizer-BioNTech BNT162b2DRPB (Table 7). The risk of death in a group equals the number of deaths from COVID-19 in an age group divided by the total number of cases in the group.\n\n* Bernal et al.54\n\n** Authorsâ calculation.\n\nStep 3: Estimate the number of people infected in Kenya expected to die from COVID-19 without vaccination PoPDwithout as a product of PoPIwithout (918,656.41) and DRunvaccinated (0.131380546). Thus, PoPDwithout=PoPIwithoutÃDRunvaccinated=918,656.41Ã0.131380546=120,693.58.\n\nStep 4: Estimate the number of people in Kenya expected to die from COVID-19 even though vaccinated with Pfizer-BioNTech BNT162b2PoPDwith through the multiplication of the number of people expected to contract COVID-19 though vaccinated PoPIwith (from Step 5 of Subsection 2.5.1) by the probability of death in a vaccinated group DRPB of 0.068. In Kenya, for instance, the PoPIwith equals 310,578.71 persons multiplied by DRPBof 0.068, i.e. PoPDwith=PoPIwithÃDRPB=310,578.71Ã0.068=21,119.35.\n\nStep 5: The number of COVID-19-associated deaths prevented COVIDâ19DPrevented, assuming 30% population vaccine coverage, equals the difference between the number of people expected to die of COVID-19 without PoPDwithout and with PoPDwith vaccination, multiplied by 30%. In other words, COVIDâ19DPrevented=PoPDwithoutâPoPDwithÃ30/100. The number of deaths averted through 30% vaccination coverage equals 29,872.27, which is 120,693.58 (from Step 3 in Subsection 2.5.2) minus 21,119.35 (from Step 5 in Subsection 2.5.2) multiplied by 0.30.\n\nStep 6: The indirect cost savings expected from vaccination equals the number of deaths prevented COVIDâ19DPrevented (Step 5 in Subsection 2.5.2) multiplied by the average indirect cost per COVID-19 death in Kenya ATICKENYA (Subsection 2.4.1 Equation 9). For example, the expected savings from COVID-19-associated deaths prevented in Kenya equals Int$1,100,277,535.56, i.e. 29,872.27 deaths prevented (among 15 years and older) multiplied by the indirect cost per death from COVID-19 of Int$36,832.74.\n\n\n3. Results\n\n3.1.1 Findings from the present value of life analysis assuming Kenyaâs average both sexes life expectancy of 67.47 years and a discount rate of 3%\n\nAs of 25 July 2022, Kenya had lost 5,670 human lives from COVID-19, translating to 66,980 UYLL, equivalent to 49,065 DYLL. As depicted in Table 8, the cumulative number of human life losses had a TPVKENYA of Int$268,408,687 and an average total present value of Int$47,338 per human life (i.e., about eight times the GDP per capita for Kenya).\n\nApproximately 3.6% of the TPVKENYA was borne by 0â9-year-olds, 2.4% by 10â19-year-olds, 7.3% by 20â29-year-olds, 17.4% by 30â39-year-olds, 21.9% by 40â49-year-olds, 22.3% by 50â59-year-olds, and 25.2% by 60âyear-olds and above. The persons between 20 and 59 yearsâthe most economically productive bracketâincurred 68.9% (Int$185,000,542) of the TPVKENYA. The TPVKENYA decreases as the age of the person advances. For instance, the 0â9-year-olds average TPV of Int$154,025 was eight-fold higher than Int$47,338 among the 60-year-olds and above.\n\n3.1.2 Share of the TPV by administrative counties in Kenya\n\nFigure 1 depicts the share of the TPVKENYA across the 47 administrative counties in Kenya.\n\nThe average TPVKENYA was Int$5,710,823 per county, with a standard deviation of Int$23,349,696. The size of TPVKENYA varied widely between counties, i.e., from a minimum of Int$14,934 (in Elgeyo Marakwet, Samburu, and West Pokot Counties) to a maximum of Int$157,926,994 in Nairobi County. Thirty-five (74.5%) counties had a TPVKENYA of less than Int$1,000,000; six counties (12.8%) had between Int1,000,000 and Int$10,000,000; four counties (8.5%) had between Int$10,000,001 and Int$20,000,000; two (4.3%) counties had over Int$20,000,000. Five counties (Kajiado, Kiambu, Machakos, Mombasa, and Nairobi City) bore 86% of the TPVKENYA. Nairobi city alone bore 58.8% (Int$157,926,994) of the TPVKENYA. The size of TPVKENYA borne by a county hinge on the number of COVID-19 life losses sustained.\n\n3.1.3 Sensitivity analysis\n\n3.1.3.1 Impact of changes in the discount rate\n\nTable 9 shows that the re-run of the HKA model with a discount rate of 5% led to a decrease in the TPVKENYA from Int$ 268,408,687 to Int$226,791,171, which is a 16% (Int$41,617,516) decrease. The ATPVKENYA decreased from Int$47,338 to Int$39,998 per COVID-19-associated death.\n\nRe-estimation of the HCA model with a 10% discount rate, all other factors held constant, reduced the TPVKENYA from Int$268,408,687 to Int$164,679,113, which was a 39% reduction (Int$103,729,574). The ATPVKENYA decreased from Int$47,338 to Int$29,044 per COVID-19-associated death.\n\n3.1.3.2 Effect of changes in life expectancy at birth\n\nAs portrayed in Table 10, a re-estimation of the economic model with Africa's highest life expectancy at birth of 78.76 years (Algeria's females) grew the TPVKENYA from Int$268,408,687 to Int$480,899,177, which is 79% (Int$212,490,490) growth. Likewise, the mean ATPVKENYA grew from Int$47,338 per human life (obtained assuming a national life expectancy of 67.47 years) to Int$84,815.\n\nRe-estimation of the economic model with the World's highest life expectancy at birth of 88.17 years (Hong Kong females) increased the TPVKENYA from Int$268,408,687 to Int$613,747,054, which is 129% (Int$345,338,367) growth. The ATPVKENYA grew from Int$47,338 per human life (obtained assuming a national life expectancy of 67.47 years) to Int$108,245.\n\n3.1.3.3 Effect of changes in the number of deaths due to COVID-19\n\nAs portrayed in Table 11, a re-run of the economic model with the excess mortality of 180,215, instead of the reported 5670 COVID-19 deaths, increased the TPVKENYA by 3,078% (Int$8,262,796,784), i.e., from Int$268,408,687 to Int$8,531,205,470.\n\n3.2.1 The indirect costs (or productivity losses) of reported deaths\n\nAs shown in Table 12, the 5586 COVID-19-reported deaths among those within the economically productive age bracket of 15 years and above resulted in a total indirect cost of Int$ 205,747,692; and an average total indirect cost per death of Int$ 36,833.\n\nAll the 84 deaths that occurred below the age of 15 years, which were not within the working age bracket, were valued at zero. Out of the total productivity losses, 0.3% were borne by 15â19-year-olds; 5.6% by 20â29-year-olds; 19.2% by 30â39-year-olds; 24.3% by 40â49-year-olds; 24.5% by 50â59-year-olds; and 26.1% by 60-year-olds and above.\n\n3.2.2 The direct cost of caring for reported COVID-19 cases\n\nAs depicted in Table 13, the estimated total direct cost of caring for the reported 330,910 cases was Int$545,401,259.29; and the average total direct cost was Int$1,648.2 per patient.\n\nOf these, 25% was for home-based isolation and care for asymptomatic cases, 22.9% for hospital/isolation centre care for mild/moderate cases, 7.4% for hospital high dependency unit care for severe cases, and 44.4% for hospital intensive care unit care for critical cases. As expected, due to the resource-intensive nature of hospital intensive unit care, the care of critically sick COVID-19 patients accounted for almost half of the total direct cost.\n\nWe estimate that the 30% target population's COVID-19 vaccination coverage may have saved Kenya a total cost of Int$ 1,400,945,809. It consists of Int$300,668,273 direct cost savings associated with the prevention of 182,423 COVID-19 projected infections and indirect cost savings of Int$1,100,277,536 from 29,872 deaths averted among 15-year-olds and above.\n\n\n4. Discussion\n\nThis study has seven key findings. First, the 5,670 human lives Kenya reported to have lost from COVID-19 had a TPVKENYA of Int$268,408,687, equivalent to 0.1% of Kenya's total GDP in 2022. Second, the ATPVKENYA of Int$47,338 per human life was eight times the per capita GDP of Kenya. Third, about 59% of TPVKENYA accrued only in Nairobi City County. Fourth, sensitivity analysis revealed that an increase in discount rate reduces TPVKENYA, increases in life expectancy at birth augment TPVKENYA, and increases in the number of deaths associated with COVID-19 grow the estimated TPVKENYA. Fifth, the 5586 COVID-19-reported deaths in the economically productive age bracket of 15 years and above resulted in a total indirect cost of Int$ 205,747,692 and an average total indirect cost per death of Int$ 36,833. Sixth, the estimated total direct cost of caring for the reported 330,910 cases was Int$545,401,259.29, and the average total direct cost was Int$1,648.2 per patient. Seventh, the 30% target population COVID-19 vaccination coverage may have saved Kenya a total cost of Int$ 1,400,945,809.\n\nAs depicted in Table 14, Kenya's ATPVKENYA was lower than all the 15 countries that also applied a similar human capital model.\n\nFor instance, the ATPVKENYA for Kenya of Int$47,338 is less than those of Spain by approximately 10-fold, Italy by 8-fold, China by 8-fold, France by 7-fold, Mauritius by 7-fold, USA by 6-fold, Japan by 6-fold, Canada by 5-fold, Turkey by 5-fold, UK by 5-fold, Germany by 4-fold, Iran by 3-fold, Brazil by 2-fold, India by 2-fold, and South Africa by 2-fold. Kenyaâs lower ATPVKENYA might be related to the lower GDP per capita3 and the lower average life expectancy at birth.48\n\nFirst, the discounted monetary values of life reported in our paper hinge on the number of COVID-19-associated deaths reported by the Government of Kenya (GoK). COVID-19 Excess Mortality Collaborators estimated that the GoK may have underestimated excess mortality due to the pandemic by 31.784-fold.49 Consequently, our TPVKENYA estimate of Int$268,408,687 might be underestimated by 31.784-fold.\n\nSecond, due to the unavailability of research resources, we could not compare our estimates using the HKA with those of alternative human life valuation methods (IVA and CVA) highlighted in the Methods section.37\n\nThird, our study uses the GDP per capita as a proxy indicator of the value the Kenyan society attaches to human statistical life. As discussed by Giannetti et al.,55 Stiglitz et al.,56 Fleurbaey57 and Kahneman and Deaton,58 the indicator is not an indicator of overall well-being (quality of life, happiness, wellness) of society as it ignores social-economic-political-ecological inequities, omits environmental costs (e.g. depletion of natural resources, global warming due to pollution), and excludes most non-monetary production (e.g. child and elderly care at home, household chores by full-time homemakers).\n\nFourth, the HKA omits a person's non-monetary value to the bereaved family,44 the psychological pain of the loss of a loved one, takes account only of society's loss in national income and ignores the person's desire to live.56\n\nFifth, our study captures only one of the adverse effects of the global COVID-19 pandemic, i.e. the associated mortality. It does not value non-fatal short-term and long-term effects on victimsâ health, which could be significant.59\n\nSixth, our study suffers the limitations explained by Baraza et al.42 because it used their direct unit cost estimates. Furthermore, in calculating direct cost, Baraza et al.42 did not consider the out-of-pocket expenses incurred by COVID-19 patients and their families and friends during diagnosis, isolation, management, and rehabilitation. Thus, in that respect, the total direct cost savings due to the COVID-19 vaccination reported in our paper might be underestimated.\n\n\n5. Conclusions\n\nThe study estimated the total present value of human lives lost in Kenya as of 25 July 2022 to be 0.1% of the national GDP. The average total present value per human life loss of Int$47,338 due to COVID-19 was eight times the per capita GDP of Kenya.\n\nThe reported COVID-19 cases cost the country an estimated total of Int$751,148,951, of which 27.4% was indirect costs (productivity losses), and 72.6% was direct costs. However, by 25 July 2022, Kenya had vaccinated 30% of the projected target population with COVID-19 vaccines, which may have saved the country a total cost of Int$ 1,400,945,809.\n\nThe pandemic continues to erode human health (quality of life and life expectancy) and economic development. However, scaling COVID-19 vaccination coverage would save Kenya substantial direct and indirect costs.\n\nTo mitigate the health and economic effects of the current and future public health emergencies, Kenya ought to augment health development investments to bridge the extant gaps in diseases surveillance system (IHR capacities),10 NHS (national and devolved),9 systems that address other basic needs,12 and national health research system.17 Furthermore, the economic evidence adduced in this paper complements arguments of human rights to life, medical care, education, clothing, food, housing, and social security when health sector policymakers are making a case for bolstering investments in health-related systems.60,61\n\n\nAuthor contributions\n\nJMK, GMM, and RNDKM contributed to the literature review, data extraction from various databases, conceptualisation, development of the economic models on Microsoft Excel Software, formal analysis, findings interpretation, and manuscript writing. All authors approved the final version of the paper.\n\n\nEthical approval and consent to participate\n\nThe study did not require ethical approval because it relied wholly on the secondary data published in international databases of the International Monetary Fund (IMF), Republic of Kenya COVID-19 statistics, Worldometers, and the World Health Organization (WHO).",
"appendix": "Data availability\n\n\n\n⢠Worldometers. https://www.worldometers.info/coronavirus/country/kenya/. 8\n\n- Covid-19 case data\n\n⢠International Monetary Fund (IMF) World Economic Outlook Database. https://www.imf.org/en/Publications/WEO/weo-database/2021/October 3.\n\n- GDP data\n\n⢠Worldometers. https://www.worldometers.info/demographics/life-expectancy/#countries-ranked-by-life-expectancy. 48\n\n- Average life expectancy data\n\n⢠World Health Organization (WHO) Global Health Expenditure Database. https://apps.who.int/nha/database/Select/Indicators/en. 19\n\n- Per capita current health expenditure data\n\n\nAcknowledgements\n\nWe are grateful to Jehovah Shalom for shielding our lives and livelihoods throughout the study. The paper is dedicated to health workers for their immense sacrifice during the ongoing fight against COVID-19. 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}
|
[
{
"id": "187248",
"date": "21 Aug 2023",
"name": "Carl AB Pearson",
"expertise": [
"Reviewer Expertise Infectious disease epidemiology",
"specializing in mathematical modelling."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall:\nThis kind of valuation work is critical to understanding the consequences of epidemics, and therefore the potential benefits attainable by general improvements in medical and public health systems. Pre-establishing these kind of assessments, and associated data sources and uncertainties, can also provide timely evidence for emergent and crisis decision-making about interventions with clear tradeoffs for health versus economic productivity.\nAt its current stage, this work seems like a useful start towards satisfying those objectives. However, there are flaws to be addressed.\nClarity / accuracy:\nWhile I appreciate the overview of Kenyan macro-economic indicators to some extent, the introduction and other elements of the manuscript sometimes feels like a laundry list of facts, many of which aren't pertinent to argument and analysis offered. The result is a bit of noisy muddle where its hard to pick out which statistics actually matter (and thus should have their provenance chased down by reviewers / readers).\nAlmost all of the values quoted in the manuscript are offered to a precision that is unlikely to be justified (I would wager there's at most 2 significant figures supported by most of these sources) and I noted no estimation intervals.\nMost of the results seem like they would more appropriately presented as plots (though I would also want to be able to download tabulated data as a spreadsheet of values).\nThe equations use a large number of variable names. Some of these are documented in table 3, some elsewhere. These would be more manageable if table 3 where presented at the outset of the methods section, and included all variables that appeared later in the equations. Table 3 also seems to reference a value for Croatia - unclear why this is.\nThe variable names also seem inordinately long, to the point of making the equations difficult to parse; most of the cases of subscript \"KENYA\" can be deleted (that values concern Kenya is implicit from context). Inclusion of units in the variable names is also strange to me, but that might be a field practice distinction. It seems like there are some variables that are always used together - I think this is the cases for COVIDD_KENYA and PD_i - these seem like an opportunity to consolidate to make the equations more accessible. In several places, the equations are written out with expanded subscripts (e.g. eq 6), but the point of subscripts is to not fully elaborate them.\nAppropriateness:\nOverall, the equations seem mathematically fine. However, I think there are some items potentially worth addressing in terms of choice of data / equation to represent phenomena.\nLife expectancy differs per birth cohort. Seems possible to use the age-specific life expectancy from Kenyan national statistics or the world population project or similar.\n\nI would expect GDP per capita to also vary by age group (e.g. the youngest workers have yet to obtain mastery of complex skills, efficient practices, etc, while the oldest workers might have lost energy and edge or be unaware of improved practices), though I can imagine that resolution might not be available in the data\n\nI am a bit surprised by no specification of a maximum productive working age, and that people continue churning out GDP until expected end-of-life. Does that accurately reflect the labor force in Kenya? I'm not assuming the pensioner lifestyles that occur in high-income settings, but it seems likely infirmity strikes before death.\n\nDoes it make sense to use the \"target\" vaccine coverage for estimating savings? I do see some value to that, but I'd definitely prefer to see the achieved coverage\n\nI'm also wary of these aggregate averted values. The vaccine did not exist for effectively the first year of the pandemic. Why does it make sense to reduce infections that happened before then?\n\nIn the other direction, vaccination general promotes reduced transmission risk. There is work that attempts to incorporate that kind of effect (e.g. http://dx.doi.org/10.1136/bmjgh-2022-009430) - perhaps it makes more sense to use output estimates from dynamical models than to crudely calculate those effects here. What the authors appear to do instead is to assume an identical attack rate in a fully unvaccinated and fully vaccinated population, use efficacy indicators to estimate the (reduced) attack rate among the fully vaccinated, and then multiple the difference of those values by the coverage.\nSufficiency of detail / Reproducibility:\nI have not attempted to replicate this study. It seems like most or all of the data and equations associated with the results are present. What would make this substantially clearer to assess (and generally improve confidence in the results), would be for the authors to share their spreadsheet (formulas + data). Per the F1000 guidelines, the authors should make their analytical code available, which means they should make those spreadsheets available for review and for readers.\nAt least some of the data sources appear retrospectively inaccessible. I definitely can't access (7) to look up the age distribution of deaths, nor (46) for labor force statistics.\nStatistical analysis:\nAs mentioned in section on clarity / accuracy - in general, values are presented to an accuracy which seems unlikely to be justified and without any attempts to use ranges. Most of these values should be used an appropriate resolution in significant figures, and the authors should be propagating uncertainty in estimates into their results.\nConclusions supported:\nTo the extent that the results represent essentially accounting and that the authors faithful executed all the math they described in their spreadsheet implementation, the numerical values are supported as mathematical outcomes. Per my previous notes, however, its not obvious to me that the results themselves are sufficiently sound to draw the non-mathematical conclusions, e.g. how the TPV of lives lost compares to the GDP lost otherwise during the pandemic.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "195293",
"date": "19 Sep 2023",
"name": "Eunice YS Chan",
"expertise": [
"Reviewer Expertise Evidence-Based Medicine",
"Health Technology Assessment",
"Health Policy",
"Public Health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article estimated the total present value of human lives lost in Kenya, total indirect costs of COVID-19 mortality, direct costs of all cases and expected savings due to COVID-19 vaccination. It added economic evidence of Kenya on COVID-19 and may potentially help the pandemic control in Kenya. Overall it is clear and easy to follow.\nFor calculating the expected savings in projected total indirect costs due to COVID-19 vaccination (section 2.5.2), the vaccine effectiveness in reducing the risk of death was based on the study âEffectiveness of the Pfizer-BioNTech and Oxford-AstraZeneca vaccines on covid-19 related symptoms, hospital admissions, and mortality in older adults in England: test negative case-control studyâ by Bernal et al,. The study investigated the effectiveness of the vaccine among adults aged above 70. However, in this study, the indirect cost or productivity loss in Kenya is the summation of indirect costs in economically productive age groups which are 15 years old and above. Are there any other studies that focus on the vaccine effectiveness of a more general population or the majority of the economically productive age groups? Using evidence that is from a similar age group as the productive age groups in Kenya may result in a more accurate estimation of the risk of death due to COVID-19 among the unvaccinated, which then can calculate a more meaningful and accurate prediction of the expected savings in projected total indirect costs.\nAlso, for expected savings in total direct costs due to COVID-19 vaccinations (2.5.1), the saving costs were estimated using the Oxford-AstraZeneca vaccine and for savings in projected total indiret costs, Pfizer-BioNTech BNT162b2 was used even though the paper by Bernal et al., did investigate the  Oxford-AstraZeneca vaccine. What are the reasons for the use of evidence of 2 different vaccines?\nThe COVID-19 vaccines require 2 doses to be considered as fully vaccinated. Due to the difficulty of delivering the 2 doses, the efficacy difference of 2 doses and 1 dose may need to be taken into consideration. Due to this, a sensitivity analysis of the coverage of vaccines can be conducted to take into account the various uptake of the population.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-232
|
https://f1000research.com/articles/12-229/v1
|
01 Mar 23
|
{
"type": "Research Article",
"title": "Validation of Depression, Anxiety and Stress Scale (DASS-21) among healthcare workers during the outbreak of delta variant of SARS-CoV-2 in Ghana",
"authors": [
"Kissinger Marfoh",
"Ali Samba",
"Eunice Okyere",
"Priscilla Kushigbor",
"Franklin Acheampong",
"Ali Samba",
"Eunice Okyere",
"Priscilla Kushigbor",
"Franklin Acheampong"
],
"abstract": "Background: Today COVID-19 is having a dire effect on the mental and physical health of the general population. Although the long-term psychological effects of COVID-19 remain unknown, studies have shown increasing depression, anxiety and stress among healthcare workers. The aim of the study was to examine the psychometric properties and validation of the Depression, Anxiety, and Stress Scale (DASS-21) and evaluate the level of depression, anxiety, and stress among healthcare workers in a tertiary hospital during the SARS-CoV-2 delta variant outbreak. Methods: Using an online questionnaire, we conducted a cross-sectional study on 1201 healthcare workers in a tertiary hospital. The validation of DASS-21 was performed by examining the factorial structure (construct validity) using confirmatory factor analysis (CFA), internal consistency (reliability), convergent validity, discriminate validity and measurement invariance. Results: Cronbach's Alpha was acceptable for depression (0.88), anxiety (0.81), and stress (0.86). CFA provides support for the three-factor oblique model with the following fit indexes: (Chi-Square Ï2/ (degrees of freedom) = 1628.5/(186), p < 0.001), comparative fit index (CFI = 0.923), Tucker-Lewis index (TLI = 0.901), standardized root mean square residual (SRMR = 0.0452) and root mean square error of approximation (RMSEA = 0.07). DASS-21 achieved both configural and metric invariance. Conclusions: Our results indicate that DASS-21 is a reliable and valid self-reporting screening tool for depression, anxiety and stress among healthcare workers. This tool is also invariant across sex, doctors, nurses, and non-clinical healthcare workers. Thus DASS-21 is an essential screening tool to identify healthcare workers at a higher risk of developing work-related mental health disorders.",
"keywords": [
":COVID-19",
"Healthcare workers",
"depression",
"Anxiety",
"Stress",
"reliability",
"validity"
],
"content": "Introduction\n\nCOVID-19 is an infectious disease caused by SARS-CoV-2. Since it was identified and isolated in Wuhan, China, COVID-19 has quickly spread to the rest of the world, leading the World Health Organization to declare the situation a pandemic. The disease has affected 500 million people, with less than 1% mortality.\n\nToday, COVID-19 is direly affecting peopleâs mental and physical health through psychological stress imposed by the disease. Studies have shown an increase in COVID-19-related psychological stress in the general population,1â3 which include depression, anxiety, stress, and post-traumatic stress.4,5 These unprecedented psychological stresses and physical risks have been more noticed among healthcare workers and their families due to COVID-19 infection. In many countries, there are considerably more COVID-19 infections among healthcare workers than among the general population.6 With healthcare workers representing less than 3% of the population, the World Health Organization reports that 14% of COVID-19 cases have been healthcare workers.\n\nHealthcare workers are routinely exposed to stress from their occupation, which is associated with more mental health complaints than others.7,8 Besides experiencing these stresses, healthcare workers are exposed to additional stress from taking care of COVID-19-infected patients, fear of being infected and infecting family members, stigmatization, and isolation. In addition, increased COVID-19 cases and deaths, increased workload, lack of socialization and recreation, and a dearth of personal protective equipment have a tremendous psychological impact on healthcare workers.9\n\nHealthcare workers have reported psychological stress during previous infectious diseases outbreaks such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), H1N1 influenza, and Ebola.10â12 For example, during the 2003 SARS outbreak, studies reported a 5â10% prevalence of moderate-to-severe post-traumatic stress and an increased high risk of depression and anxiety among healthcare workers who were quarantined or frontline.3,13 In the 2015 outbreak of Ebola in Sierra Leone, there was 48.0% anxiety or depression and 76.0% post-traumatic stress among healthcare workers.14\n\nAlthough the long-term psychological effects of COVID-19 remain unknown, studies on healthcare workers have shown 50.4% depression, 44.6% anxiety, and 71.5% stress in China15; 24.7% depression, 44.6% anxiety and 19.8% stress in Italy16; and 11.4% depression, 23.4% anxiety and 5.7% stress in India.17 A systematic review and meta-analysis showed a high prevalence of moderate-to-severe depression, anxiety and stress among healthcare workers during the COVID-19 pandemic.18 In Ghana, healthcare workers reported an increased prevalence of depression, anxiety, and stress during the early phase of the COVID-19 pandemic.19 Furthermore, studies comparing emergency and non-emergency healthcare workers found that emergency healthcare workers had a high prevalence of depression, anxiety and stress.20,21\n\nWith the poor quality of patient care,22 the rising number of medical errors and medical litigation,23 and increased disability and absenteeism due to increased levels of poor mental health among healthcare workers, it has become important to screen for mental health issues among healthcare workers. One of the most commonly used scales for screening mental health is the Depression, Anxiety and Stress Scale (DASS).24 Since it was developed in 1995, the psychometric properties of DASS and its short version, DASS-21 (21 items), have been evaluated in both clinical25 and non-clinical26 samples for validity and reliability. The self-reporting scale has 42 items (DASS-42), which was reduced to 21 to form the short version (DASS-21). Seven items with the highest factor loadings from each subscale of the original DASS were selected to form the DASS-21. DASS-21 has three main subscales: depression, anxiety and stress. The depression scale evaluates hopelessness, self-deprecation, dysphoria, devaluation of life, lack of interest/involvement, anhedonia and inertia. The anxiety scale evaluates autonomic arousal, skeletal muscle effects, situational anxiety, and subjective experience of anxious affect. The stress scale evaluates nervous arousal, difficulty relaxing and being easily upset and impatient.\n\nMany previous studies have reported excellent reliability and internal consistency of the DASS-21 in healthcare workers.4 However, there has been some inconsistency in the factorial structure of DASS-21. This has led to the development of other structural models apart from the original three-factor model. The one-factor model developed by Lovibond and Lovibond24 has shown a good fit in many studies. A two-factor structure of DASS-21 was the best fit in the Brazilian adolescent study.27 However, in the general population, a tripartite structural model consisting of anhedonia, physiology, hyperarousal, and general negativity affect fit better than other proposed DASS-21 structures. The bifactor model (quadripartite) has also been a good fit in several studies.\n\nDASS-21 has been used extensively in various clinical settings and research because of its ability to differentiate between depression distinctly, anxiety and stress clusters of symptoms.24,26,28 DASS-21 is designed to detect the symptoms of depression, anxiety and stress. In the clinical setting, DASS-21 cannot be used to diagnose people with depression or anxiety. DASS-21 alerts the doctor to identify people who are highly susceptible to clinical depression or anxiety, indicating the need for further investigations.24\n\nDASS-21 has been validated for different populations, but, to date, no research paper has previously validated the psychometric properties of DASS-21 in a Ghanaian population. This has led to its limited use as a screening tool for mental health; therefore, it is critical to evaluate the psychometric properties of DASS-21 among healthcare workers. This study aimed to examine the psychometric properties of the DASS-21 scale and evaluate the level of depression, anxiety, and stress among healthcare workers in a tertiary hospital during the SARS-CoV-2 delta variant outbreak in Ghana.\n\n\nMethod\n\nWe conducted a cross-sectional study to determine the psychometric property of DASS-21 among healthcare workers in Korle-Bu Teaching Hospital. This study used an online survey method and recruited participants using a convenience sampling technique. Healthcare workers were invited to complete an online survey (https://kbth.gov.gh/kbredcap/) which was distributed through the hospitalâs social media platforms. The online survey, created by the research electronic data capture (REDCap) system (RRID:SCR_003445),29,30 was open for two months (June 3âAug 3 2021). The survey was aimed at all healthcare workers in Korle-Bu Teaching Hospital working in various departments. These included doctors, nurses, administrators, record officers, pharmacists, laboratory scientists, etc.\n\nThe inclusion criteria for the study were all healthcare workers who were at the post during the outbreak of the delta variant. Healthcare workers on leave, travelling, ill, and those with known mental health disorders were excluded from the study. A healthcare worker was defined as anyone employed by the Ministry of Health to work at Korle-Bu Teaching Hospital.\n\nThe authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. The Korle-bu Teaching Hospital institutional review boards (IRD: KBTH-ADM/00014/2021) in Ghana approved all study procedures on 2nd February 2021. Electronic informed consent was obtained from all participants.\n\nWe estimated the minimum sample size based on one item to 10 participants.31 Therefore, the minimum acceptable sample size based on 21 items of DASS-21 was 210 respondents. However, the total number of healthcare workers participating in the study was 1201.\n\nThis study was conducted during the hospitalâs SARS-CoV-2 delta variant outbreak. The online survey sent to the participants consists of an introductory statement and an electronic informed consent that briefly explained the studyâs aims, purpose, and benefit. It also informed participants that their information would be used for research purposes, kept confidential, and would not be shared with the hospitalâs management. After reading the introductory statement, participants who selected âyesâ meant that they had consented to participate in the study and were allowed to answer questions in the demographic and eligibility section. Only those who met the inclusion criteria answered the rest of the questionnaire. All answers to the questions were required; thus, there were no missing observations. In total, 1201 healthcare workers were enrolled in the study. All data collected were anonymous and kept confidential.\n\nDuring the study period, the global incidence of confirmed COVID-19 cases had increased by 25%. In Ghana, the average weekly cases increased from 45 to 2000. Similarly, the total number of confirmed cases in the hospital increased from 5 to 80 per week.\n\nDemographic variables\n\nDemographic variables were obtained from a self-reported online questionnaire that included age, sex duration of work, occupation or profession and working in an emergency unit.\n\nDepression, Anxiety and Stress Scale\n\nDASS-21 assesses the state of depression, anxiety, and stress in the last week. DASS-21 consists of three subscales with seven items, each assessing depression, anxiety, and stress. It uses a 4-point Likert response for each item from 0 to 3 (0 = âdid not apply to me at allâ to 3 = âapplies to me most of the timeâ). A subscale score is generated by summing relevant responses. The composite score of each subscale was multiplied by two (therefore making DASS-21 (short version) comparable to the DASS-42 scale (original version). A high composite score indicates a higher psychological stress.24,28\n\nAll statistical analyses were performed using SPSS (version 26) (RRID:SCR_002865), but the confirmatory factor analysis (CFA) was performed using AMOS (version 26) (RRID:SCR_022686. Descriptive statistics used in this paper included mean and standard deviation, and proportion and percentage.\n\nWe first validated the DASS-21 by examining the factorial structure (construct validity), internal consistency, convergent validity, and discriminate validity. Validation assesses the ability of the DASS-21 questionnaire to measure the latent construct (depression, anxiety, and stress) intended to be measured. After validation, we examined measurement invariance across the sex and professional groups. Before validation analysis, KaiserâMeyerâOlkin (KMO) and Bartlettâs tests were performed to check the suitability of the dataset. The results indicated that our sample was adequate for the CFA (Chi-square Ï2 (210) = 13064.5, p < 0.01) and the overall measurement of sampling adequacy was 0.95.\n\nCFA with maximum likelihood estimation was performed to determine the structure and item loading in DASS-21. We tested five models of DASS-21, which included a one-factor model in which all the 21 items load on a general distress factor,24 a two-factor model in which stress and anxiety were combined as one factor and depression as another factor,32 the original three-factor model,24 a bifactor model with general distress (negatively affectivity) as a factor, with depression, anxiety and stress as specific factors26 and a tripartite model in which all items load on a negative affectivity factor, and a specific factor that is made up of anxiety and depression.33\n\nThe results from CFA were analyzed based on the following fit indexes and goodness-of-fit criteria: 1) adequate fit: root-mean-square error (RMSE) (<0.08),34,35 comparative fit index (CFI) (>0.9)36; and 2) good fit: RMSE (<0.05)35 and CFI (>0.95).36 The Ï2 test result is strongly dependent on the sample size but it is estimated for all models.\n\nThe internal consistency of the DASS-21 and its subscales (Depression, Anxiety and Stress) were assessed using Cronbachâs alpha. A cut-off Cronbach alpha value of > 0.7 indicates high internal consistency.37,38 Pearsonâs correlation coefficient was used to evaluate the item-total correlation. Items with a very low correlation coefficient of < 0.3 were deleted.37\n\nWe assessed both the convergent and discriminant validity of the best-fit model. Convergent validity is the ability of the items to successfully estimate the latent variable(s) of the best-fit model. The average variance extracted (AVE) and composite reliability (CR) were used to assess the convergent validity of the best-fit model. For convergent validity to be significant, a CR value of 7 or greater (CR > 7) and AVE values of 0.5 or greater are required.39\n\nDiscriminant validity determines how a model can distinctly identify depression, anxiety and stress from other factors. We assessed discriminant validity by comparing the amount of variance explained by the construct and the share variance with other constructs. Thus, for a model to have discriminate validity, the square root of the AVE of the measured construct should be larger than the correlation coefficients of the other constructs.40\n\nA multi-group CFA with sex and professional group was performed to evaluate measurement invariance. The first was the configural model, in which all parameters were unrestricted across the group, and then the metric model, where there was factor loading to equality between groups. Metric invariance was evaluated as a function of a difference between the unconstrained and constrained models. The lack of invariance was indicated when the Ï2 test of goodness of fit was significant (p < 0.05). Since Ï2 is sample-size dependent, we also considered a model to be invariant across a group with a slight change (â) in CFI and root mean square error of approximation (RMSEA)âCFI and RMSEA should not exceed 0.02 and 0.015, respectively.41\n\n\nResults\n\nA total of 1201 healthcare workers participated in the study. The mean age of the participants was 34 years (SD = 7.1 years), ranging from 20 to 58 years. The participants were predominantly females, nurses, and had worked for less than three years (53.9%). One-fifth of the participants worked in emergency-related units. DASS-21 provided an independent assessment of depression (mean = 13.6, SD = 2.7), anxiety (mean = 15.8, SD = 3.1) and stress score (mean = 12.8, SD = 3.3) with mean scores indicating mild depression, severe anxiety and normal stress levels (Table 1).\n\nThe summary of the fit statistics is shown in Table 2, indicating that the one-factor and two-factor models had unacceptable fit values. The fit values for the bifactor and tripartite models provided an improved and acceptable fit. However, the three-factor model (Figure 1) provided the best fit for the data.\n\nThe standardized factor loadings indicated higher and excellent factor loadings for depression, anxiety and stress, except item 2 (âDryness of the monthâ) with a factor loading of 0.38. Therefore, item 2 was considered for removal. The final three-factor model (Figure 2) was further enhanced by allowing correlation between items from the same subscale using a high modification index of > 10. The modified DASS-21 provided the final best-fit model to data (CFI: 0.961, Tucker-Lewis index (TLI): 0.929, standardized root mean square residual (SRMR): 0.032, RMSEA: 0.071).\n\nFrom Table 3, the Cronbach alpha for the total DASS-21 scale was adequate. Internal consistency for the three subdomain scales was very good: DASS-21 depression (DASS21-D) was 0.88, DASS-21 anxiety (DASS21-A) was 0.86, and DASS-21 stress (DASS21-S) was 0.81. The item scale and total item scale correlations were satisfactory and ranged from 0.3 to 0.7 and 0.4 to 0.8, respectively.\n\nWe estimated the convergent validity based on the value of CR and average variance extracted. From Table 4, all the constructs (depression, anxiety and stress) met the predefined criteria for convergent validityâCR > 0.7, CR > AVE and AVE > 0.5. For discriminant validity, the square of the average variance extracted for depression (0.726), anxiety (0.713) and stress (0.707) constructs were all higher than their correlation with other constructs (off-diagonal) (Table 4). This indicates that depression, anxiety, and stress are distinct elements (discriminant validity), as proposed by Lovibond and Lovibond.25 All constructs passed the convergent and discriminant validity tests.\n\nThe multi-group CFA was used to evaluate the degree of measurement invariance of DASS-21 across sex and professional group. Table 5 shows that the configural and metric models for both sex and professional group provided an excellent fit for the data. The invariance test indicates equivalence across males and females. The increase in model constraint reveals no significant difference between the configural and metric (Ï2 (17) = 21.1, p > 0.05). For the professional group, configural invariance shows that the data fit adequately to the model without any constraint. The difference between the metric and configural invariance was not statistically significant, which indicates that metric invariance was achieved.\n\nThe prevalence of moderate-to- severe symptoms of depression, anxiety and stress among healthcare workers was 14.2% (170), 4.5% (54) and 30.8% (370), respectively (Table 6). Doctors had high psychological stress compared with other healthcare workers. Healthcare workers who worked in the emergency setting or for more than 10 years had a higher prevalence of depression, anxiety, and stress than others. Moderate-to-severe depression, anxiety, and stress symptoms were equivalent across males and females. However, healthcare workers older than 50 years had lower psychological stress incidence (Table 6).\n\n\nDiscussion\n\nTo our knowledge, this study is the first to examine the psychometric properties of DASS-21 and provide evidence for its use as a measure of mental health issues among Ghanaian healthcare workers. Overall, our results support the three-dimension structures of DASS-21 as reported in previous studies.24,28,42 This indicates that DASS-21 can be used as a self-reported tool to assess mental health issues of depression, anxiety, and stress among healthcare workers.\n\nThe construct validity of DASS-21 was satisfactory based on the CFA, and all model fit indexes were acceptable. Our CFA showed that the best-fit model for our data was the three-factor oblique model for depression, anxiety, and stress, which has been previously validated in clinical and non-clinical samples.25,26,28,43 The three-factor oblique model was better than the one-factor and two-factor models, indicating that depression, anxiety and stress are distinct constructs, as previously reported.24,44\n\nA modification improved model fitness to items with poor factor loadings (factor loading < 0.4). However, item 2 (âdryness of the mouthâ) from the anxiety subscale shows weaker factor loadings than other items in our study sample. Previous studies have questioned the utility of item 2 (âdryness of the mouthâ) as a measure of the anxiety construct.25,45â48 On the contrary, some studies have suggested that item 2 (âdryness of the mouthâ) performed excellently in the psychological stress construct (one-factor model).45,47 Moreover, Parkitny et al.46 described the âdryness of the mouthâ item as a vague response to DASS-21. Besides the initial improvement, the three-factor model was further enhanced using a modification index that allows correlation error between items from the same scale.\n\nOur reliability results for DASS-21 showed an excellent internal consistency for all the subscales, with the Cronbach alpha ranging from 0.81 to 0.93. These results confirmed previous reports about the reliability of DASS-21 in different studies and populations.26,28,49,50 We confirm from previous studies that DASS-21 has a high convergent and discriminant validity of the three constructs.26,42,49,51\n\nIn this study, we also examined invariant measurement across sex and professional groups. Our results showed that DASS-21 has a full configural and metric invariance across sex and professional groups. This indicates that the meaning of DASS -21 to measure depression, anxiety, and stress was the same for females and males or doctors, nurses, and non-clinical healthcare workersâthere were no differences across sex and professional groups in the DASS-21 structure.\n\nIn our study, the prevalence of depression, anxiety, and stress among healthcare workers was 14.4% (DASS-21 depression score > 13), 30.8% (DASS-21 anxiety score > 9) and 4.5% (DASS-21 stress score > 18), respectively. These prevalences of depression, anxiety and stress were lower than those reported in previous studies of Ghanaian healthcare workers during the early phase of the pandemic19 and other studies from China,15 Italy,16 and India.17 The differences in the prevalence of depression, anxiety, and stress may be due to sociocultural differences affecting the individualâs experience and expression of mental health symptoms. This shows that DASS-21 can be used as a screening tool to identify healthcare workers with depression, anxiety and stress. The tool is relevant for the hospital employee assistance program, which aims to identify healthcare workers with mental health issues in order to provide support for them.\n\nThe high prevalence of depression and anxiety reported among healthcare workers may be due to the possible impact of the delta variant of SARS-CoV-2 in the hospital. During the COVID-19 outbreak, especially in the vaccine-availability era, when there was widespread disease and an increased number of deaths, it was very common to observe high psychological stress among healthcare workers who may have directly or indirectly managed COVID-19-infected patients. Similar events of the high prevalence of mental health issues among healthcare workers have been observed in the SARS, MERS, H1N1 influenza, and Ebola outbreaks.\n\nWith the advent of the COVID-19 vaccine, healthcare workersâ mental status depends on the vaccineâs ability to prevent them from being infected and infecting others. The decrease in vaccine efficacy resulting in increased breakthrough infections severely impacts their mental and physical health. Moreover, the virulent nature of the delta variant, the high influx of infected patients with severe clinical manifestations or hospitalization, high mortality, increased workload, fear of getting infected and the dearth of personal protective equipment all negatively affect the mental health status of healthcare workers.\n\nThe strength of this study is that it is the first time a DASS-21 validation has been performed among Ghanaian healthcare workers. In addition, this present study provides novel information about the prevalence of mental health issues among healthcare workers during the delta variant outbreak. One limitation of this study was the self-reported response of DASS-21, which may have led to potential biases or measurement errors. For example, some healthcare workers may have provided a socially acceptable response about their mental health issues because of stigmatization. Additionally, using a convenient sampling method could result in selection bias because only healthcare workers with internet access completed the online questionnaire. Finally, all the respondents were healthcare workers from the same hospital, which may affect the generalization of the findings.\n\n\nConclusions\n\nIn summary, our results indicate that DASS-21 has good psychometric properties among Ghanaian healthcare workers. We found DASS-21 to be valid and reliable, with the ability to distinctly identify depression, anxiety, and stress from other factors. Our data suggest that this tool is invariant across sex and across doctors, nurses, and non-clinical healthcare workers. Thus, DASS-21 can be used as a self-reporting tool to screen for depression, anxiety, and stress in health workers. This will allow authorities, such as employee assistance programs, to identify healthcare workers at higher risk of developing work-related mental health disorders.",
"appendix": "Data availability\n\nFigshare: Dataset for psychometric evaluation of depression, anxiety, and stress scale among healthcare worker in Ghana during COVID-19 outbreak. Dataset. https://doi.org/10.6084/m9.figshare.21929697. 52\n\nThis project contains the following underlying data:\n\n- dass4_3.csv\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nCénat JM, Blais-Rochette C, Kokou-Kpolou CK, et al.: Prevalence of symptoms of depression, anxiety, insomnia, posttraumatic stress disorder, and psychological distress among populations affected by the COVID-19 pandemic: A systematic review and meta-analysis. Psychiatry Res. 2021; 295: 113599. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVindegaard N, Benros ME: COVID-19 pandemic and mental health consequences: Systematic review of the current evidence. Brain Behav. Immun. 2020; 89: 531â542. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu T, Jia X, Shi H, et al.: Prevalence of mental health problems during the COVID-19 pandemic: A systematic review and meta-analysis. J. Affect. Disord. 2021; 281: 91â98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen J, Liu X, Wang D, et al.: Risk factors for depression and anxiety in healthcare workers deployed during the COVID-19 outbreak in China. Soc. Psychiatry Psychiatr. Epidemiol. 2021; 56(1): 47â55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpoorthy MS, Pratapa SK, Mahant S: Mental health problems faced by healthcare workers due to the COVID-19 pandemicâA review. Asian J. Psychiatr. 2020; 51: 102119. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Keep health workers safe to keep patients safe.2020.\n\nDubey S, Biswas P, Ghosh R, et al.: Psychosocial impact of COVID-19. Diabetes Metab. Syndr. Clin. Res. Rev. 2020; 14(5): 779â788. Publisher Full Text\n\nMcAlonan GM, Lee AM, Cheung V, et al.: Immediate and sustained psychological impact of an emerging infectious disease outbreak on health care workers. Can. J. Psychiatry. 2007; 52(4): 241â247. PubMed Abstract | Publisher Full Text\n\nAlkhamees AA, Alrashed SA, Alzunaydi AA, et al.: The psychological impact of COVID-19 pandemic on the general population of Saudi Arabia. Compr. Psychiatry. 2020; 102: 152192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrace SL, Hershenfield K, Robertson E, et al.: The occupational and psychosocial impact of SARS on academic physicians in three affected hospitals. Psychosomatics. 2005; 46(5): 385â391. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSultan S, Bashar A, Nomani I, et al.: Impact of COVID-19 pandemic on psychological health of a sample of the health care workers in the western region of Kingdom of Saudi Arabia. Middle East Curr. Psychiatry. 2022; 29(1): 1â11. Publisher Full Text\n\nTam CW, Pang EP, Lam LC, et al.: Severe acute respiratory syndrome (SARS) in Hong Kong in 2003: stress and psychological impact among frontline healthcare workers. Psychol. Med. 2004; 34(7): 1197â1204. Publisher Full Text\n\nBai Y, Lin C-C, Lin C-Y, et al.: Survey of stress reactions among health care workers involved with the SARS outbreak. Psychiatr. Serv. 2004; 55(9): 1055â1057. PubMed Abstract | Publisher Full Text\n\nJalloh MF, Li W, Bunnell RE, et al.: Impact of Ebola experiences and risk perceptions on mental health in Sierra Leone, July 2015. BMJ Glob. Health. 2018; 3(2): e000471. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLai J, Ma S, Wang Y, et al.: Factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019. JAMA Netw. Open. 2020; 3(3): e203976âe203976. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRossi R, Socci V, Pacitti F, et al.: Mental health outcomes among frontline and second-line health care workers during the coronavirus disease 2019 (COVID-19) pandemic in Italy. JAMA Netw. Open. 2020; 3(5): e2010185âe2010185. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElbay RY, KurtulmuÅ A, ArpacıoÄlu S, et al.: Depression, anxiety, stress levels of physicians and associated factors in Covid-19 pandemics. Psychiatry Res. 2020; 290: 113130. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarvaldi M, Mallet J, Dubertret C, et al.: Anxiety, depression, trauma-related, and sleep disorders among healthcare workers during the COVID-19 pandemic: A systematic review and meta-analysis. Neurosci. Biobehav. Rev. 2021; 126: 252â264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOfori AA, Osarfo J, Agbeno EK, et al.: Psychological impact of COVID-19 on health workers in Ghana: A multicentre, cross-sectional study. SAGE Open Med. 2021; 9: 205031212110009. Publisher Full Text\n\nThâng F, Rao KA, Ge L, et al.: A One-Year Longitudinal Study: Changes in Depression and Anxiety in Frontline Emergency Department Healthcare Workers in the COVID-19 Pandemic. Int. J. Environ. Res. Public Health. 2021; 18(21): 11228. PubMed Abstract | Publisher Full Text | Free Full Text\n\nÄ°lhan B, KÃŒpeli Ä°: Secondary traumatic stress, anxiety, and depression among emergency healthcare workers in the middle of the COVID-19 outbreak: A cross-sectional study. Am. J. Emerg. Med. 2022; 52: 99â104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson J, Louch G, Dunning A, et al.: Burnout mediates the association between depression and patient safety perceptions: A cross-sectional study in hospital nurses. J. Adv. Nurs. 2017; 73(7): 1667â1680. PubMed Abstract | Publisher Full Text\n\nKakemam E, Kalhor R, Khakdel Z, et al.: Occupational stress and cognitive failure of nurses and associations with self-reported adverse events: A national cross-sectional survey. J. Adv. Nurs. 2019; 75(12): 3609â3618. PubMed Abstract | Publisher Full Text\n\nLovibond S, Lovibond P: Manual for the depression anxiety stress scales. Sydney. Nsw:Psychology Foundation of Australia;1995.\n\nClara IP, Cox BJ, Enns MW: Confirmatory factor analysis of the DepressionâAnxietyâStress Scales in depressed and anxious patients. J. Psychopathol. Behav. Assess. 2001; 23(1): 61â67. Publisher Full Text\n\nHenry JD, Crawford JR: The short-form version of the Depression Anxiety Stress Scales (DASS-21): Construct validity and normative data in a large non-clinical sample. Br. J. Clin. Psychol. 2005; 44(2): 227â239. Publisher Full Text\n\nSilva HA, Passos MHP, Oliveira VMA, et al.: Short version of the Depression Anxiety Stress Scale-21: is it valid for Brazilian adolescents? Einstein (São Paulo). 2016; 14: 486â493. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAntony MM, Bieling PJ, Cox BJ, et al.: Psychometric properties of the 42-item and 21-item versions of the Depression Anxiety Stress Scales in clinical groups and a community sample. Psychol. Assess. 1998; 10(2): 176â181. Publisher Full Text\n\nHarris PA, Taylor R, Minor BL, et al.: The REDCap consortium: Building an international community of software platform partners. J. Biomed. Inform. 2019; 95: 103208. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarris PA, Taylor R, Thielke R, et al.: A metadata-driven methodology and workflow process for providing translational research informatics support. J. Biomed. Inform. 2009; 42(2): 377â381. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShoukri MM, Asyali M, Donner A: Sample size requirements for the design of reliability study: review and new results. Stat. Methods Med. Res. 2004; 13(4): 251â271. Publisher Full Text\n\nBrown TA, Chorpita BF, Korotitsch W, et al.: Psychometric properties of the Depression Anxiety Stress Scales (DASS) in clinical samples. Behav. Res. Ther. 1997; 35(1): 79â89. Publisher Full Text\n\nTully PJ, Zajac IT, Venning AJ: The structure of anxiety and depression in a normative sample of younger and older Australian adolescents. J. Abnorm. Child Psychol. 2009; 37(5): 717â726. PubMed Abstract | Publisher Full Text\n\nAwang Z: Structural equation modeling using AMOS graphic. Penerbit Universiti Teknologi MARA;2012.\n\nLt H, Bentler PM: Cutoff criteria for fit indexes in covariance structure analysis: Conventional criteria versus new alternatives. Struct. Equ. Model. Multidiscip. J. 1999; 6(1): 1â55.\n\nBentler PM: Comparative fit indexes in structural models. Psychol. Bull. 1990; 107(2): 238â246. Publisher Full Text\n\nNunnally JC: Psychometric theory 3E. Tata McGraw-Hill Education;1994.\n\nDeVellis RF, Thorpe CT: Scale development: Theory and applications. Sage publications;2021.\n\nHair J Jr, Black W, Babin B, et al.: Canonical correlation. Suplemento de Multivariate Data Analysis. 7th Edition.Pearson;2014. Reference Source\n\nFornell C, Bookstein FL: Two structural equation models: LISREL and PLS applied to consumer exit-voice theory. J. Mark. Res. 1982; 19(4): 440â452. Publisher Full Text\n\nAli AM, Hendawy AO, Ahmad O, et al.: The Arabic version of the Cohen perceived stress scale: Factorial validity and measurement invariance. Brain Sci. 2021; 11(4): 419. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNorton PJ: Depression Anxiety and Stress Scales (DASS-21): Psychometric analysis across four racial groups. Anxiety Stress Coping. 2007; 20(3): 253â265. PubMed Abstract | Publisher Full Text\n\nKyriazos TA, Stalikas A, Prassa K, et al.: Can the Depression Anxiety Stress Scales Short be shorter? Factor structure and measurement invariance of DASS-21 and DASS-9 in a Greek, non-clinical sample. Psychology. 2018; 09(5): 1095â1127. Publisher Full Text\n\nWatson D, Clark LA: Negative affectivity: the disposition to experience aversive emotional states. Psychol. Bull. 1984; 96(3): 465â490. PubMed Abstract | Publisher Full Text\n\nLe MTH, Tran TD, Holton S, et al.: Reliability, convergent validity and factor structure of the DASS-21 in a sample of Vietnamese adolescents. PLoS One. 2017; 12(7): e0180557. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParkitny L, McAuley JH, Walton D, et al.: Rasch analysis supports the use of the depression, anxiety, and stress scales to measure mood in groups but not in individuals with chronic low back pain. J. Clin. Epidemiol. 2012; 65(2): 189â198. Publisher Full Text\n\nSzabó M: The short version of the Depression Anxiety Stress Scales (DASS-21): Factor structure in a young adolescent sample. J. Adolesc. 2010; 33(1): 1â8. PubMed Abstract | Publisher Full Text\n\nShea TL, Tennant A, Pallant JF: Rasch model analysis of the Depression, Anxiety and Stress Scales (DASS). BMC Psychiatry. 2009; 9(1): 1â10. Publisher Full Text\n\nBados A, Solanas A, Andrés R: Psychometric properties of the Spanish version of depression, anxiety and stress scales (DASS). Psicothema. 2005; 679â683.\n\nOsman A, Wong JL, Bagge CL, et al.: The depression anxiety stress Scalesâ21 (DASS-21): further examination of dimensions, scale reliability, and correlates. J. Clin. Psychol. 2012; 68(12): 1322â1338. PubMed Abstract | Publisher Full Text\n\nTonsing KN: Psychometric properties and validation of Nepali version of the Depression Anxiety Stress Scales (DASS-21). Asian J. Psychiatr. 2014; 8: 63â66. PubMed Abstract | Publisher Full Text\n\nMarfoh M, Samba A, Eunice Okyere E, et al.:Validation of Depression, Anxiety and Stress Scale (DASS-21) among healthcare workers during the outbreak of delta variant of SARS-CoV-2 in Ghana. Dataset. Figshare. 2023. Publisher Full Text"
}
|
[
{
"id": "180287",
"date": "27 Jun 2023",
"name": "Roy Rillera Marzo",
"expertise": [
"Reviewer Expertise Epidemiology",
"Health Promotion",
"Research Methodology",
"Communicable and Non-Communicable Diseases",
"Environmental Health",
"Global Sustainable Development",
"Health and Social Services",
"Nutrition",
"Ageing and Quality of Life",
"Social and Behavioural Health",
"Healthcare Management",
"Strategic Management",
"Health Services Management",
"Health Leadership Management",
"Quality Management",
"Medical Education"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe introduction of your manuscript titled \"Validation of Depression, Anxiety and Stress Scale (DASS-21) among healthcare workers during the outbreak of delta variant of SARS-CoV-2 in Ghana\" is detailed and provides an in-depth background of the study. However, here are a few suggestions for improvement:\nClarity and specificity: The current manuscript lacks a clear and concise explanation of the specific context of the study (i.e., the Delta variant). I would suggest emphasizing how the Delta variant has specifically impacted healthcare workers' mental health in Ghana, perhaps necessitating a re-evaluation or adjustment of the DASS-21 for this unique context.\n\nLength: The introduction is quite lengthy. While it is rich in information, the reader could potentially get lost in the details. Consider pruning less critical information or consolidating points to make it more reader-friendly.\n\nConsistency: Some figures provided appear inconsistent, especially in the part where the number of COVID-19 cases among healthcare workers is mentioned. Please ensure that your data is consistent and correctly cited.\n\nMethods:\nSampling strategy: Although convenience sampling can be a practical method, it's crucial to mention its limitations. Convenience samples may not be representative of the entire healthcare worker population at Korle-Bu Teaching Hospital, introducing a potential bias that could limit the generalizability of your findings. Consider discussing this aspect in the limitations of the study.\n\nSelf-reported data: The methodology relies heavily on self-reported data, which can introduce the risk of response bias, including social desirability bias and recall bias. Discussing these concerns in your methods section would contribute to a more balanced view of your results.\n\nInclusion and Exclusion criteria: You excluded healthcare workers with known mental health disorders. However, this exclusion could lead to an underestimation of the prevalence of stress, anxiety, and depression, as those with pre-existing mental health conditions may be more likely to experience these symptoms.\n\nSurvey design: More information on the design of the questionnaire might be helpful. For instance, was the DASS-21 questionnaire modified in any way for this study? Also, was it translated? If so, did it go through a rigorous translation-back translation process to ensure it retains its meaning?\n\nData cleaning: It might be worth mentioning if any data cleaning procedures were performed before analysis, as some responses might have been inconsistent or illogical.\n\nIncidence of COVID-19: You provided data on the incidence of COVID-19 during the study period. How was this information collected and verified? Detailing this process could add more credibility to your study.\n\nConfounding variables: The role of potential confounding variables and how these were controlled for in the study should be stated. For example, demographic variables such as age, sex, duration of work, occupation or profession, and working in an emergency unit might influence the psychometric properties of DASS-21.\n\nMeasurement invariance: You've tested measurement invariance across sex and professional groups. Did you consider other subgroups, such as age groups or levels of exposure to COVID-19 patients? This could provide more insights into the instrument's performance across different subpopulations.\n\nStatistical analysis: The methodology would benefit from more information about how missing data were handled in your analyses. Although you mentioned that all answers to the questions were required, it is still possible that some participants did not complete the survey fully.\n\nDiscussion:\nComparison with other studies: Lack of comparison to other countries. The previous studies (9 references listed below) can help. Although you've compared the prevalence of depression, anxiety, and stress in your study with other countries, it might be beneficial to discuss why these differences might exist. For instance, are there certain factors unique to Ghanaian healthcare workers that could explain the lower prevalence rates?\n\nItem 2 on the DASS-21: You've discussed the controversy regarding the \"dryness of the mouth\" item. However, it would be helpful to provide more context. How does this item's weak performance in your study contribute to this ongoing debate? Does this imply that this item may not be as relevant in the context of Ghanaian healthcare workers?\n\nImplications for Practice: The application of the findings to practical scenarios, such as how it might influence policy or guide future research, needs more emphasis. How can healthcare organizations use this information to better support their employees?\n\nStrengths and Limitations section:\nSampling Strategy: Your admission of potential selection bias due to the convenience sampling method and online survey is important. However, it may also be worthwhile to discuss ways you could mitigate these issues in future studies, such as using stratified or random sampling techniques.\n\nSingle Site Study: As the study was conducted in one hospital, the findings may not be applicable to other settings in Ghana or elsewhere. Future studies could aim to include multiple sites for a more comprehensive understanding.\n\nCross-Sectional Design: As a cross-sectional study, it captures the participants' mental health status at one point in time. It would be beneficial to acknowledge that this design doesn't allow for conclusions about cause-and-effect relationships or changes over time.\nConclusion section:\nClaims About DASS-21 Applicability: Although DASS-21 demonstrated good psychometric properties in this study, it may be an overstatement to say it can be used as a screening tool in \"health workers.\" This study focused on healthcare workers at a specific hospital in Ghana, and the results may not be directly applicable to health workers in different settings or countries.\n\nFuture Research Directions: The conclusion would benefit from a call to action for future research. How can other researchers build upon your findings? This might involve testing DASS-21 in a wider population of Ghanaian healthcare workers or comparing its performance with other mental health screening tools.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-229
|
https://f1000research.com/articles/10-1120/v1
|
08 Nov 21
|
{
"type": "Clinical Practice Article",
"title": "New onset asthma during pregnancy: two case reports",
"authors": [
"Paula S. Schriek",
"Saar A. Bendien",
"Hanneke A. Feitsma",
"Jeroen van Exsel",
"Saar A. Bendien",
"Hanneke A. Feitsma",
"Jeroen van Exsel"
],
"abstract": "Introduction: Asthma is the most common chronic respiratory disease during pregnancy. However, reports of new onset asthma during pregnancy are lacking. We report two cases of new onset asthma during pregnancy following respiratory tract infection, subsequently one case with M. pneumoniae infection and the other case with a combined infection with respiratory syncytial virus and rhinovirus.\n\nCase presentation:\n\nBoth patients presented with the clinical features of an acute asthma exacerbation during pregnancy without a medical history of asthma. During follow up the diagnosis of asthma was supported by spirometry showing significant reversibility and elevated fractional exhaled nitric oxide (FeNO). Patients were hospitalized and received supplemental oxygen, treatment for an acute asthma exacerbation with systemic corticosteroids, high dose inhalation therapy. These therapeutic interventions subsequently led to a good outcome for the mother and newborn in both cases.\n\nConclusions: New onset asthma should be part of the differential diagnosis in pregnant patients with respiratory symptoms, particularly in case of mycoplasma infection. Diagnosing asthma during pregnancy can be challenging. In these circumstances, additional diagnostic tests (like inflammatory biomarkers FeNO and blood eosinophils) can be helpful to support the diagnosis.",
"keywords": [
"asthma",
"pregnancy",
"infection",
"exacerbation"
],
"content": "Abbreviations\n\nb.i.d., twice a day; body mass index, BMI; coronavirus disease 2019, COVID-19; C-Reactive Protein test, CRP; Estimated fetal weight, EFW; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; FENO, fractional exhaled nitric oxide; gestational age, GA; immunoglobulin M, IgM; inhaled corticosteroid, ICS; OCS, oral corticosteroids; respiratory syncytial virus, RSV; severe acute respiratory syndrome coronavirus, SARS-CoV-2\n\n\nIntroduction\n\nAsthma is one of the most common chronic diseases during pregnancy with a prevalence of 7â10%. Dyspnoea is a frequent symptom, being present in 60â70% of healthy pregnancies, mainly as a result of normal physiological changes (Table 1)1. When a patient presents with dyspnoea during pregnancy it can be challenging to distinguish dyspnoea related to physiological changes (such as decrease in functional residual capacity and pregnancy-induced hyperventilation (Table 1.)) from pathology, such as uncontrolled asthma. This can be particularly difficult in patients without a medical history of asthma. Reports of new onset asthma during pregnancy are lacking. Uncontrolled asthma and asthma exacerbations during pregnancy are associated with increased risk of adverse outcomes for mother and child such as preterm birth, low birth weight and pre-eclampsia2,3. Viral respiratory infections are the most common trigger for asthma exacerbations. M. pneumoniae infection is also known to play a role in asthma, resulting in exacerbations, and possibly related to the pathogenesis of asthma4,5. It is thought that pregnant women are more susceptible to infections or that they are more severely affected by infectious diseases6,7. In this report, we describe two patients with de novo diagnosis of asthma during pregnancy following respiratory tract infection.\n\n\nCase reports\n\nA healthy 33-year-old Caucasian woman presented to the emergency department with progressive dyspnoea in her third trimester of pregnancy, at 32 weeks gestation. The general practitioner treated her with clarithromycin, salbutamol aerosol 100 ÎŒg/do and oral corticosteroids (OCS) (prednisone 30mg) and beclomethasone 100 ÎŒg/do metered-dose inhaler twice daily (b.i.d.). Despite this therapy, her clinical condition deteriorated with progressive work of breathing and cough.\n\nHer medical history consisted of scoliosis and one episode of possible pneumonia, treated by her primary care physician with antibiotics. Independent of this event, she never experienced respiratory symptoms. She had no family history of asthma and no personal history of atopy. She stopped smoking 5 years previously. Her body mass index (BMI) was 24 mg/m2. She was employed as a teacher.\n\nAt presentation, the patient was in moderate respiratory distress. Vital signs showed an oxygen saturation of 98% on room air and blood pressure of 130/70mmHg.\n\nLung auscultation revealed an expiratory wheeze with scattered rhonchi. The physical examination and chest radiograph showed no abnormalities. The white-blood-cell count was 16.3Ã109/L, with neutrophils of 15.3Ã109/L, and eosinophils of 0.05 Ã109/L. C-Reactive Protein test (CRP) was low (4 mg/L). Arterial blood gas analysis showed respiratory alkalosis with pH 7.46, pCO2 3.6 kPa, HCO3 â 21 mmol/L, pO2 11.6kPA, with an elevated A-a gradient of 3.9 kPa.\n\nSputum cultures showed upper respiratory tract flora. Serum immunoglobulin M (IgM) antibody titer was 16 for M. pneumoniae, which is indicative of current infection. Allergy testing with Phadiatop test was negative (Total serum IgE: 33 IU/ml).\n\nThe patient was admitted to the hospital due to respiratory distress and treated with prednisone 30mg/day for 10 days, clarithromycin was increased to 500 mg b.i.d. in combination with salbutamol/ipratropium 1/0.2 mg/ml inhalation solution every four hours. The beclomethasone inhaler was converted to a high dose inhaled corticosteroid and a long-acting beta-agonist (ICS/LABA) (beclomethasone/formoterol aerosol 200/6 µg, two inhalations b.i.d.). Throughout the course of the week, the patient remained tachypnoeic, oxygen dependent and frequent nebulizing was needed. After a week, her symptoms improved and OCS was systematically tapered. Daily cardiotocography and biweekly fetal growth ultrasound were normal, with an estimated fetal weight (EFW) of p57 and normal amniotic fluid. The patient was discharged after a hospital stay of 15 days.\n\n\nFollow-up and outcomes\n\nTwo weeks after discharge, and discontinuation of OCS, spirometry revealed FVC 3.37L (84%), FEV1 2.75L (82%) pre bronchodilator (pre-BD), FEV1 3.12L (93%) post bronchodilator (post-BD). Spirometry demonstrates significant reversibility (13% and 262 cc) which is compatible with asthma. Furthermore, strongly elevated fractional exhaled nitric oxide (FeNO) (60ppb) and elevated blood eosinophils levels (0.50Ã109/L) were found. Due to persistent eosinophilic inflammation, treatment with ICS was intensified. FeNO normalized after 8 weeks (18ppb). The outcomes after pregnancy were remarkably good for the patient and newborn. No maternal complications during labor or late term pregnancy occurred. The patient gave birth to a healthy baby boy at a GA of 39 weeks and three days, with a birthweight of 3445 grams. The baby had an uncomplicated neonatal course. Over a period of 6 months, inhalation medication could be reduced to ICS only and the patient was referred back to primary care.\n\nA 22-year-old Turkish woman, with morbid obesity (BMI of 46) and no further medical history, presented to the emergency department with dyspnoea and a productive cough in her first trimester of pregnancy, at 8 weeks gestation. She developed a cold with nasal congestion, a productive cough and progressive dyspnoea. There was no family history of asthma and she had never smoked. She was employed as a housekeeper.\n\nAt presentation, vital signs showed a respiratory rate of 23 breaths/minute, oxygen saturation of 92% on room air and blood pressure of 135/85. Lung auscultation revealed an extended expirium with scattered rhonchi. The chest radiograph showed no signs of pneumonia. The white-blood-cell count was 12.6Ã109/L, with neutrophils of 10.0Ã109/L and eosinophils of 0.28Ã109/L. CRP was elevated; 76 mg/L. Arterial blood gas analysis showed hypoxemia and respiratory alkalosis with pH 7.44, pCO2 4.0 kPa, HCO3 22 mmol/L, pO2 8.4 kPa, with an elevated A-a gradient of 6.6 kPa.\n\nThe patient was admitted to the hospital. Treatment was started with prednisone 30 mg/day, clarithromycin 500 mg b.i.d. in combination with salbutamol/ipratropium 1/0.2 mg/ml nebulizer and inhaled oxygen. High dose beclomethasone/formoterol aerosol 200/6 µg was started with two inhalations b.i.d. Polymerase chain reaction on throat swab was positive for respiratory syncytial virus (RSV) and rhinovirus. Phadiatop test was positive with increased IgE for dog rose, house dust mite, timothy grass and birch pollen (total serum IgE level 1632 /IU/ml).\n\nSpirometry during hospitalization revealed FEV1 2.47L (78%) and FEV1/FVC (84.31%), Z-score of -0.68. The patient gradually recovered, and antibiotics, prednisone and salbutamol/ipratropium inhalation were discontinued after three days. The patient was discharged after six days.\n\nOne month after discharge she returned to our emergency department with complaints of a common cold with a sore throat, a productive cough and dyspnoea. At presentation, the patient was not clinically in distress and there was no hypoxemia. Treatment was started with prednisone 30 mg/day for seven days and the patient was discharged. Spirometry performed in the outpatient setting revealed FEV1 1.81L (57%) pre-BD, FEV1 2.07L (66%) post-BD, FEV1/FVC (70.29%, Z-score: -2.53). Spirometry demonstrates significant reversibility (15% and 260 cc) which supports the diagnosis of asthma. Furthermore, FeNO was strongly elevated (58ppb). At 20 weeks gestational age (GA), spirometry revealed FEV1 3.25L (103%) and FEV1/FVC (86.44%, Z-score: -0.32), demonstrating significant variability over time.\n\nThe patient developed gestational diabetes mellitus in the third trimester. Monthly growth ultrasound showed normal fetal growth (EFW p72), normal abdominal circumference (fetal abdominal circumference p44) and normal amniotic fluid.\n\n\nFollow-up and outcomes\n\nAt 28 weeks, the patient presented to our emergency department again with dyspnoea and a non-productive cough. She was clinically stable with an adequate oxygen saturation of 97%. The blood eosinophil count was 0.36Ã109/L. with a CRP of 23 mg/L. Newly diagnosed was an iron deficiency anaemia (haemoglobin: 5.6 mmol/L) which recovered after ferricarboxymaltose 1000mg (haemoglobin afterwards 7.6mmol/L). She was admitted to the hospital and treated with prednisone 30 mg/day for five days and salbutamol/ipratropium 1/0.2 mg/ml inhalation. Both sputum cultures and serum IgM for M. pneumoniae were\n\nDuring admission, daily fetal assessment was performed, ultrasound scan of fetal growth was normal. The patient was discharged after seven days. the pregnancy outcomes of the patient were normal. She gave (uncomplicated vaginal) birth to a healthy baby girl at a GA of 41+4 with a birthweight of 3805 grams. The apgar score was 9-10-10 after 1,5 and 10 minutes.\n\nIn the outpatient setting the patient received two more follow-ups from the respiratory physician and the specialized respiratory nurse. During these consultations no more asthma exacerbations were reported but unfortunately the patientâs adherence to medication seemed poor. After these consultations the patient was lost to follow up in secondary care. Further monitoring of asthma will be performed by primary care.\n\n\nDiscussion\n\nNew onset asthma during pregnancy may not always be recognized but might be rather common. Based on pulmonary function testing, inflammatory biomarkers and clinical response to treatment, our patients are strongly suspected to have new onset asthma triggered by infection during pregnancy. To the best of our knowledge, there are no previous case reports about new onset asthma during pregnancy following mycoplasma infection. Both patients presented with severe exacerbations requiring hospitalization, oxygen supplementation and treatment with OCS and antibiotics. These interventions subsequently led to a good outcome for the mother and newborn in both cases. This illustrates the importance of active treatment of asthma exacerbations during pregnancy.\n\nThe diagnosis of asthma during pregnancy can be challenging as bronchoprovocation testing with metacholine or histamine is contra indicated during pregnancy8. Healthy pregnant women show no change in FEV1 and FVC during (late) pregnancy. So, in patients suspected of new onset asthma during pregnancy, and normal spirometry, repeating spirometry when the patient is having more symptoms (as in case 2) to demonstrate significant variability or additional inflammatory biomarkers (as in case 1) can support the diagnosis. This is important since over- as well as under-treatment should be avoided in pregnant patients with asthma.\n\nIn non-pregnant women an exacerbation, which can be triggered by viral infections, may be the presenting manifestation of asthma. Mycoplasma infection resulting in new onset, infection- mediated, asthma, outside pregnancy, is also well recognized4. These two phenomena are not as well described in pregnancy. Current literature suggests that M. pneumoniae is an important trigger for acute exacerbations of asthma, accounting for 3.3â50% of all exacerbations. However, the role of M. pneumonia infection in the pathogenesis of asthma is still controversial5,9. In 1994, Yano et al., reported a case of mycoplasma infection leading to a first presentation of asthma, suggesting the infection led to a complex interplay of airway inflammation and IgE mediated hypersensitivity (type 1 reaction as seen in asthma)10. Recent studies also show that M. Pneumoniae infection in non-pregnant women and children can contribute to the development of asthma and prevalence of asthma is increased in patients with a history of atypical pneumonia1,4,5,11. Although these findings are suggestive of an association between M. pneumoniae and asthma disease onset and exacerbations, larger well controlled studies are needed to test this hypothesis and identify underlying mechanisms.\n\nThe role of other viral respiratory tract infections, especially RSV or rhinovirus, in the development of asthma is also a subject of interest. Certain components seem to contribute to the risk of developing asthma, such as type and severity of viral infections and host characteristics12. It is well known that pregnant women are more susceptible to viral infections and that they are more at risk for developing severe diseases. For pregnant women with asthma this risk is even higher13\n\nPregnancy is a pro-inflammatory and immune modulating condition, depending on the stage of pregnancy6,7,14,15. Theoretical, physiological immunosuppression is a characteristic of pregnancy and results in feto-maternal tolerance, although this is a complex interacting network and not yet fully understood. Changes in immune characteristics during pregnancy including a shift in (Th1)/Th2 ratio toward a Th2- predominant immune state and an increase in regulatory T cells (Tregs) may explain increased susceptibility to infections16,17. These alterations in immune function possibly also have a role in triggering the process of developing new onset asthma during pregnancy.\n\nThe current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic that caused the coronavirus disease 2019 (COVID-19), resulted in many deaths worldwide. Recent studies suggest that pregnancy is associated with increased risk for severe illness due to the SARS-CoV-2 infection. Pregnant patients with COVID-19 are more likely to need intensive care unit admission, invasive ventilation and there is an increased risk for preterm birth18â20. The ongoing discussion whether or not asthma should be considered a risk factor for severe COVID-19 is not yet closed21. Nevertheless, despite critical gaps in our current knowledge, pregnant patients with asthma and COVID-19 should be managed by a multidisciplinary approach to optimize maternal and neonatal outcomes22. Moreover, offering vaccination with one of the two messenger RNA (mRNA) vaccines to all pregnant and breastfeeding women is recently recommended23.\n\nA limitation of these case reports is their relatively short duration of follow-up and the fact that describing these cases cannot lead to conclusions regarding causality. Furthermore, no previous data and diagnostic asthma tests were available, so we cannot fully exclude a diagnosis of asthma prior to these pregnancies. However, based on the extensive medical history of both patients this seems unlikely.\n\nIn summary, new onset asthma should be part of the differential diagnosis of pregnant patients with respiratory symptoms, especially when mycoplasma infection is found. Uncontrolled asthma during pregnancy triggered by infection is associated with adverse pregnancy outcomes in mother and child. This should alert clinicians to seriously evaluate dyspnoea during pregnancy by physical examination, repetitive spirometry and inflammatory biomarkers such as FeNO and blood eosinophils.\n\nMore studies are needed to determine the relation between respiratory tract infections and new onset asthma during pregnancy.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of the patientsâ clinical details was obtained from both patients.\n\nThis research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.",
"appendix": "References\n\nBidad K, Heidarnazhad H, Pourpak Z, et al.: Frequency of asthma as the cause of dyspnea in pregnancy. Int J Gynaecol Obstet. 2010; 111(2): 140â3. PubMed Abstract | Publisher Full Text\n\nAbdullah K, Zhu J, Gershon A, et al.: Effect of asthma exacerbation during pregnancy in women with asthma: a population-based cohort study. Eur Respir J. 2020; 55(2): 1901335. PubMed Abstract | Publisher Full Text\n\nBaghlaf H, Spence AR, Czuzoj-Shulman N, et al.: Pregnancy outcomes among women with asthma. J Matern Fetal Neonatal Med. 2019; 32(8): 1325â1331. PubMed Abstract | Publisher Full Text\n\nYeh JJ, Wang YC, Hsu WH, et al.: Incident asthma and Mycoplasma pneumoniae: A nationwide cohort study. J Allergy Clin Immunol. 2016; 137(4): 1017â1023.e6. PubMed Abstract | Publisher Full Text\n\nSutherland ER, Martin RJ: Asthma and atypical bacterial infection. Chest. 2007; 132(6): 1962â6. PubMed Abstract | Publisher Full Text\n\nRacicot K, Kwon JY, Aldo P, et al.: Understanding the complexity of the immune system during pregnancy. Am J Reprod Immunol. 2014; 72(2): 107â16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMor G, Aldo P, Alvero AB: The unique immunological and microbial aspects of pregnancy. Nat Rev Immunol. 2017; 17(8): 469â482. PubMed Abstract | Publisher Full Text\n\nGlobal initiative for asthma: Global strategy for asthma management and prevention. 2019; [cited 2020]. Reference Source\n\nNisar N, Guleria R, Kumar S, et al.: Mycoplasma pneumoniae and its role in asthma. Postgrad Med J. 2007; 83(976): 100â4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYano T, Ichikawa Y, Komatu S, et al.: Association of Mycoplasma pneumoniae antigen with initial onset of bronchial asthma. Am J Respir Crit Care Med. 1994; 149(5): 1348â53. PubMed Abstract | Publisher Full Text\n\nBiscardi S, Lorrot M, Marc E, et al.: Mycoplasma pneumoniae and asthma in children. Clin Infect Dis. 2004; 38(10): 1341â6. PubMed Abstract | Publisher Full Text\n\nSaglani S: Viral infections and the development of asthma in children. Ther Adv Infect Dis. 2013; 1(4): 139â50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVieira AC, Pite H, Morais-Almeida M: Asthma and pregnancy in the 2020 decade: still a matter of concern. J Matern Fetal Neonatal Med. 2021; 1â7. PubMed Abstract | Publisher Full Text\n\nHadfield KA, McCracken SA, Ashton AW, et al.: Regulated suppression of NF-κB throughout pregnancy maintains a favourable cytokine environment necessary for pregnancy success. J Reprod Immunol. 2011; 89(1): 1â9. PubMed Abstract | Publisher Full Text\n\nMor G, Abrahams V: Immunology of implantation. Immunology and Allergy Clinics of North America. In: Arici A, editor. Philadelphia: W.B. Saunders Company, 2002; 22(3): 545â565. Publisher Full Text\n\nVanders RL, Gibson PG, Wark PAB, et al.: Alterations in inflammatory, antiviral and regulatory cytokine responses in peripheral blood mononuclear cells from pregnant women with asthma. Respirology. 2013; 18(5): 827â33. PubMed Abstract | Publisher Full Text\n\nTamasi L, Horvath I, Bohacs A, et al.: Asthma in pregnancy--immunological changes and clinical management. Respir Med. 2011; 105(2): 159â64. PubMed Abstract | Publisher Full Text\n\nAllotey J, Stallings E, Bonet M, et al.: Clinical manifestations, risk factors, and maternal and perinatal outcomes of coronavirus disease 2019 in pregnancy: living systematic review and meta-analysis. BMJ. 2020; 370: m3320. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBadr DA, Mattern J, Carlin A, et al.: Are clinical outcomes worse for pregnant women at â¥20 weeks' gestation infected with coronavirus disease 2019? A multicenter case-control study with propensity score matching. Am J Obstet Gynecol. 2020; 223(5): 764â768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZambrano LD, Ellington S, Strid P, et al.: Update: Characteristics of Symptomatic Women of Reproductive Age with Laboratory-Confirmed SARS-CoV-2 Infection by Pregnancy Status - United States, January 22-October 3, 2020. MMWR Morb Mortal Wkly Rep. 2020; 69(44): 1641â1647. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBousquet J, Jutel M, Akdis CA, et al.: ARIA-EAACI statement on asthma and COVID-19 (June 2, 2020). Allergy. 2021; 76(3): 689â697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartinez FD: Asthma in the Time of COVID-19. Am J Respir Crit Care Med. 2021; 203(7): 785â786. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartins I, Louwen F, Ayres-de-Campos D, et al.: EBCOG position statement on COVID-19 vaccination for pregnant and breastfeeding women. Eur J Obstet Gynecol Reprod Biol. 2021; 262: 256â258. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "99613",
"date": "24 Nov 2021",
"name": "Dominick Shaw",
"expertise": [
"Reviewer Expertise Asthma"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes two interesting case reports where pregnant women develop features consistent with asthma. Features described include wheeze, variable FEV1 and breathlessness. Certainly, both presentations may be caused by new onset asthma but it's difficult to exclude the contribution of respiratory infection as both patients had evidence of respiratory pathogens.\nAirway infection, particularly with viruses, is well known to cause airway hyperresponsiveness (AHR). AHR can occur for several months after initial infection and is associated with similar symptoms to asthma. Some authors argue that airway hyperresponsiveness alone is enough to make a diagnosis of asthma, but other purists may argue that asthma is a combination of airway inflammation and inflammation. Either way it would be interesting to know whether symptoms persist following treatment, or resolution, of the underlying infection. Other factors pointing towards asthma would include an elevated blood eosinophil count and degree of reversibility in FEV1.\nSome factors make a diagnosis of asthma less likely-both cases had normal FEV1/FVC ratios (which reflect the effect on lung function of a gravid uterus); initial response to high dose oral Prednisolone was poor in the first case and lastly asthma symptoms seemed to improve after delivery.\nBoth these cases nicely demonstrate the difficulty of making an asthma diagnosis and the physiological effects of a gravid uterus on both asthma and normal respiratory physiology.\n\nIs the background of the casesâ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Partly",
"responses": [
{
"c_id": "7682",
"date": "13 Jan 2022",
"name": "Paula Schriek",
"role": "Author Response",
"response": "Dear Dominick Shaw, We would like to thank the reviewer for her time and critical evaluation of our manuscript entitled âNew onset asthma during pregnancy: two case reportsâ. We also appreciate the time and effort you have dedicated to providing insightful feedback on ways to strengthen our paper. We have incorporated changes that reflect the detailed suggestions you have provided. To facilitate your review of our revisions, the following is a point-by-point response to the questions and comments delivered in your e-mail dated 2021-11-24. On behalf of all authors, Sincerely, Paula Schriek HAGA Teaching Hospital Department of Respiratory medicine          Els Borst-Eilersplein 275 2545 AA The Hague, The Netherlands Phone no.: (0031) 702104392, (0031) 639592319 Fax no.: (0031) 702102150 E-mail: p.schriek@hagaziekenhuis.nl Reviewer 1 Airway infection, particularly with viruses, is well known to cause airway hyperresponsiveness (AHR). AHR can occur for several months after initial infection and is associated with similar symptoms to asthma. Some authors argue that airway hyperresponsiveness alone is enough to make a diagnosis of asthma, but other purists may argue that asthma is a combination of airway inflammation and inflammation. Either way it would be interesting to know whether symptoms persist following treatment, or resolution, of the underlying infection. Other factors pointing towards asthma would include an elevated blood eosinophil count and degree of reversibility in FEV1. We fully agree the importance of a clear confirmation of the diagnosis of asthma. Especially as we are well aware of the under- and over-diagnosis of asthma worldwide. (Aaron SD et al. Underdiagnosis and Overdiagnosis of Asthma. Am J Respir Crit Care Med. 2018 Oct 15;198(8):1012-1020) Based on our clinical experience with patients with asthma and pregnancy (working in a centre of excellence for severe asthma and our asthma-pregnancy outpatient clinic with a monthly multidisciplinary meeting for pregnant asthmatic patients), we believe these 2 patients have asthma and not âsolelyâ post viral AHR. We agree it would be more convincing to support this by additional objective parameters. Unfortunately we were not able to perform these additional diagnostic tests or perform a longer follow-up in both patients. However we think your consideration of AHR is of value to add to our considerations in the paper. We added this to the discussion section in the manuscript. Some factors make a diagnosis of asthma less likely-both cases had normal FEV1/FVC ratios (which reflect the effect on lung function of a gravid uterus); initial response to high dose oral Prednisolone was poor in the first case and lastly asthma symptoms seemed to improve after delivery. We are not sure if we understood this comment as FEV1/FVC ratio is unaffected during the course of pregnancy (references mentioned below). In the first case spirometry demonstrates significant reversibility, strongly elevated FeNO and elevated blood eosinophils levels which is compatible with asthma. In the second case spirometry demonstrates variability of FEV1, FEV1/FVC ratio (one time reduced with Z-score of -2.53), significant reversibility, strongly elevated FENO and blood eosinophils of 0.36Ã109/L under ICS, which is also compatible with asthma. References: 1. Weinberger SE, Weiss ST, Cohen WR, et al. . Pregnancy and the lung. Am Rev Respir Dis 1980; 121: 559â581  2. Jensen D, Webb KA, Davies GA, et al. . Mechanical ventilatory constraints during incremental cycle exercise in human pregnancy: implications for respiratory sensation. J Physiol 2008; 586: 4735â4750. 3. Jensen D, Duffin J, Lam YM, et al. . Physiological mechanisms of hyperventilation during human pregnancy. Respir Physiol Neurobiol 2008; 161: 76â86 4. McAuliffe F, Kametas N, Costello J, et al. . Respiratory function in singleton and twin pregnancy. BJOG 2002; 109: 765â769 5. Grindheim G, Toska K, Estensen ME, et al. . Changes in pulmonary function during pregnancy: a longitudinal cohort study. BJOG 2012; 119: 94â101 Revision of Discussion (paragraph 7, line 3-7) A limitation of these case reports is their relatively short duration of follow-up and the fact that describing these cases cannot lead to conclusions regarding causality. Furthermore, airway infection, particularly with viruses (like RSV or rhinovirus), may also cause airway hyperresponsiveness (AHR), although in the cases described in this report other biomarkers as FeNO and blood eosinophils are more compatible with the diagnosis of asthma(24). The exact role of viral respiratory tract infections in the onset or progression of asthma remains a subject of debate and in this setting should be considered on a case by case basis . Lastly, no previous data and diagnostic asthma tests were available, so we cannot fully exclude a diagnosis of asthma prior to these pregnancies. However, based on the extensive medical history of both patients this seems unlikely. 24.      Empey DW, Laitinen LA, Jacobs L, Gold WM, Nadel JA. Mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection. Am Rev Respir Dis. 1976;113(2):131-9."
}
]
}
] | 1
|
https://f1000research.com/articles/10-1120
|
https://f1000research.com/articles/11-1563/v1
|
22 Dec 22
|
{
"type": "Research Article",
"title": "Goji berry (Lycium barbarum) inhibits the proliferation, adhesion, and migration of oral cancer cells by inhibiting the ERK, AKT, and CyclinD cell signaling pathways: an in-vitro study",
"authors": [
"Amee Sanghavi",
"Ananth Srivatsa",
"Divya Adiga",
"Aditi Chopra",
"Richard Lobo",
"Shama Prasada Kabekkodu",
"Shivaprasada Gadag",
"Usha Nayak",
"Karthik Sivaraman",
"Ashmeet Shah",
"Amee Sanghavi",
"Ananth Srivatsa",
"Divya Adiga",
"Richard Lobo",
"Shama Prasada Kabekkodu",
"Shivaprasada Gadag",
"Usha Nayak",
"Karthik Sivaraman",
"Ashmeet Shah"
],
"abstract": "Background: Lycium barbarum (L. barbarum), popularly referred to as Goji berry, is a promising herb known for its powerful anti-antioxidant, antibacterial, and anti-inflammatory properties. It is used in traditional Chinese medicine due to its powerful antioxidant, antibacterial, and anti-inflammatory properties. It has also shown good anti-cancer properties and has been tested against liver, colon, prostate, breast, and cervical cancers. However, no study has yet evaluated the role of goji berries against oral cancer. Hence, the present paper aims to evaluate the anticancer properties of L. barbarum against oral squamous cell carcinoma.\nMethod: Ethanolic extract of L. barbarum (EELB) was tested for its anticancer properties by performing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, colony formation, cell proliferation, and scratch wound test. The impact of EELB on the signaling transduction pathways of Extracellular signal-regulated kinase (ERK1/2), protein kinase (AKT1), cyclin D1 and epithelial-mesenchymal transition (EMT) was also assessed by western blot. Results: The results showed that EELB can impede CAL-27 cell growth, proliferation and migration in-vitro.. It even reduced the phosphorylation of ERK1/2 and AKT1 with concomitant downregulation of cyclin D1 (CCND1), cadherin 2 (CDH2), and vimentin (VIM) and upregulation of cadherin 1 (CDH1) expression suggesting its anti-proliferative and anti-EMT effects in oral cancer. Conclusion: Goji berry has good antiproliferative and anti-invasive properties. It affects potential EMT markers and signaling transduction pathways involved in oral cancers. Hence goji berry can be tried as a potential anticancer agent to manage oral squamous cell carcinoma.",
"keywords": [
"Nutrition",
"Food",
"Fruit",
"Functional food",
"Traditional Chinese Medicine",
"Cancer",
"Oral Cancer",
"Goji Berry",
"Herbal",
"Lycium barbarum",
"Oral squamous cell carcinoma"
],
"content": "1. Introduction\n\nOral cancer is a malignant neoplasm commonly affecting the buccal mucosa, lip, gingiva, palate, tongue, and floor of the mouth.1,2 It is considered the sixth most common cancer worldwide.1 Among all the oral cancers, oral squamous cell carcinoma (OSCC) is the most common variant.3â5 According to the World Health Organization (WHO), approximately 6,57,000 patients suffer from oral and throat cancer every year, of which more than 330,000 patients die annually. The mortality is higher among the central and south Asian countries. This is because of the high use of tobacco and arecanut/betel chewing.3,4 Apart from tobacco, use of beedis andcigars, low socioeconomic status, high alcohol consumption, poor oral hygiene, poor diet, and increased incidence of Human Papilloma Virus (HPV), syphilis, and chronic candidiasis are some of the other risk factors that increase the prevalence of oral cancer in southeast Asian countries.6â8\n\nOral cancers are managed by radiation, chemotherapy, and surgery.9 However, these treatment options often deteriorate the quality of life and have a high risk of post-treatment morbidity. Patients on long-term chemotherapeutic agents often report chronic fatigue, hair loss, easy bruising, onset of infection, anemia, nausea, vomiting, loss of appetite, constipation, diarrhea, renal dysfunction, changes in libido, heart problems, reduced lung capacity and difficulty in breathing, bone and joint problems.10 The relapse of cancer cells, development of resistance to chemotherapy, and toxic effects on the other healthy tissues have also been reported with extensive use of chemotherapy. Owing to these problems, researchers are constantly exploring newer alternatives to manage cancer.\n\nPlant-derived phytochemicals and derivatives have gained tremendous popularity in managing various cancerous lesions, including oral cancer.11 Several compounds derived from plants are being used for managing various types of cancer. The herbal medicine or phytomedicine market is likely to reach $550 billion by the end of 2030.11â17 Natural products have been tried as adjuncts to conventional chemotherapeutic agents and have been âdocumented to decrease the lethal effect of using high doses of chemotherapeutic agents, and this in turn improves the quality of life of cancer and overall survival rate of patientsâ.16 A study reported that around half (50%) of anticancer drugs that were approved from the year 1940 to 2014 were natural products or were directly procured from plants.14 Another study also reported that more than 60% of patients suffering from cancer used plant-based products instead of the conventional chemotherapeutic agent.6 Talib et al. (2020) stated that âproducts derived from natural sources, such as plant-derived products; are more accessible, less expensive and toxic compared to those produced syntheticallyâ. Moreover, natural products have multiple compounds that act by varied mechanisms to inhibit the growth of cancer cells and may also prevent the onset of drug resistance.17 Many recent studies have found that when natural compounds are used along with conventional chemotherapic agents and radiation therapy, they can sensitize the tumors and have a synergistic effect.11â18 A combination therapy may enhance the therapeutic efficacy and reduces the required effective doses of chemotherapuetic agent.18â20 Many plant-derived products like allicin, curcumin, cinnamon, saffron, reservatol, shagol, etc. have been tested for managing oral cancer.21 Recently, L.barbarum, commonly known as goji berry; Chinese wolfberry; Himalayan goji, and Tibetan goji has emerged as a promising herb with anticancer, anti-inflammatory, and antioxidant properties.22â25 However, its effect in managing oral cancer has not been reported previously.\n\nL. barbarum belongs to the family âSolanaceaeâ. The plant is native to southeast European and Asian countries. It is commonly used as a dry fruit in the Himalayan, China, and Tibetan regions. It is also used as a traditional Chinese medicine.22,24 L. barbarum fruit contains abundant polysaccharides (L. barbarum polysaccharides or (LBPs), scopoletin, vitamin C analog (glucopyranosyl, L, ascorbic acid analogs), βeta-carotene, zeaxanthin, and flavonoids that have promising antioxidant, immuno-modulating and anticancer properties.24â26 Clinical and preclinical studies have confirmed the medicinal and therapeutic effects of L. barbarum for managing chronic fatigue, aging, stroke, ulcerative colitis, glaucoma, diabetes mellitus, and Alzheimer's disease. L. barbarum extract has shown promising results against cancers of the breast, liver, leukemia, colon, rectum, prostate, and cervix.21â31 Mao et al. (2011) observed that around 200 to 1,000 mg/l of polysaccharide in goji berry, known as lycium barbarum polysacchride (LBP) can inhibit the proliferation of the cancer of the colon by inducing G0/G1 arrest.29 Shen and Du (2012) also confirmed that LBP has antiproliferative effects against breast cancer cells and can arrest the cell cycle at S-phase.30 Although the antiproliferative and anticancer properties of L. barbarum are gaining popularity; to the best of our knowledge, studies evaluating the anticancer efficacy of L. barbarum against oral squamous cell carcinoma (OSCC) have not been reported. Hence, this paper aims to evaluate the anticancer effect of L. barbarum on the human OSCC cell line (CAL-27).\n\n\n2. Methods\n\nThe study is an in-vitro analysis of anticancer properties using the CAL-27 cell lines (RRID:CVCL-1107, ATCC, USA). The study was conducted from 9th July 2019 to 8th July 2020. The study was done after obtaining the institutional ethics committee approval (IEC no: 460/2019). Goji berry (Lycium barbarum L) was obtained from China's official distributor (Kenny delights Pvt. limited). Berry authentication was done by Gopal Krishna Bhat, a retired taxonomist of âPoornaprajna Collegeâ at Udupi, Karnataka, India. The sample was authenticated and the berries were deposited at the âManipal College of Pharmaceutical Sciences, Department of Pharmacognosy with voucher ref no: PP626.\n\nThe fruits (berries) of L. barbarum were first thoroughly cleaned with distilled water and then dried using a hot air oven (at 45oC). The berries were then grounded and powdered using a crusher to obtain a dried powder. Around 250 minced powder of the berries was subsequently macerated with 1000 mL of ethanol solution for three days and they were occasionally shaken to allow the ethanol to mix with the powder. The macerated solution was then strained, and the prepared solvent was evaporated via a rota evaporator. The extract prepared was brown in color, sticky and semisolid in consistency, and had a fruity fragrance. The extract was collected and stored in a desiccator30 (Figure 1).\n\n2.2.1 Cell culture\n\nThe OSCC cell line (CAL-27, RRID: CVCL-1107, ATCC, USA) was used for all the experiments. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% of fetal bovine serum (Himedia, India) at a temperature of 37°C in 5% CO2. The adherent cell monolayers were grown to 70%â80% confluency and then the cells were harvested using trypsin solution (0.25%, Himedia, India) before the experimentation. The EELB extract dissolved in dimethyl sulfoxide (DMSO) was used in all the experiments. The cells treated with DMSO (0.1%) were considered as vehicle control and the cell cultured in a complete medium without EELB extract served as control.\n\n2.2.2 Cell viability assay\n\n3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was performed to determine the effect of ELLB on the growth and division of cancer cells.32 The CAL-27 cells were first seeded at a concentration of around 5000 cells per well and were allowed to attach overnight. The cells were then treated with the extract at varying concentrations (1 ÎŒg, 10 ÎŒg, 100 ÎŒg per mL) for 48 hours. Cells treated with DMSO (0.1%) were considered as vehicle control. At the end of 48 hours, the cells were exposed for 4 hours to MTT (5mg/mL in phosphate buffer saline (PBS), Sigma Aldrich, USA). Following incubation with MTT, the media was aspirated, the formazan crystals were dissolved in DMSO solution, and the absorbance was measured at 570nm using a microplate reader (Varioskan, ThermoFischer Scientific, USA). The percentage of cell viability was calculated using the optical density and the background-corrected absorbance as follows: Percentage of cell viability = 100 X (Optical density of test group/Optical density of the control group). The data were represented as the mean±SD of the percentage of cell viability.\n\n2.2.3 Anchorage-dependent colony formation assay\n\nThe colony formation assay helps to check the viability of the cell based on the ability of a single cell to form colonies.1 The colony-forming test is considered a gold standard assay to check the survival rate of cells upon the use of an anti-cancer agent. The experiment was performed using CAL-27 cells.31 In brief, 100 cells were seeded in a 60mm cell culture petri dish and were exposed to 100 ÎŒg/mL of EELB. The media was replaced once in three days with a complete medium containing 100 ÎŒg/mL of EELB. The experiment was terminated at the end of 14 days. The cells which were treated with DMSO (0.1%) were used as vehicle control. At the end of 14 days, the media was discarded, and the cells were washed with PBS and stained for five minutes with 0.5% crystal violet dissolved in methanol. The excess stain was removed and cells were rinsed again with PBS. Following this, the cells were photographed using a gel documentation system. The experiment was performed in duplicates and repeated thrice.\n\n2.2.4 Cell migration assay\n\nCell migration assay is used to measure the capability of a cancer cell to migrate, which is indirectly linked to its potency to invade the connective tissue and metastasize. The effect of EELB on cell migration of CAL-27 was evaluated by performing the wound healing assay.33,34 CAL-27 cells were first seeded in a 6-well plate (1x105 cells per well) and the cells were left to form a monolayer. A sterile microtip was used to make scratches in the cell monolayer. After making scratches, a fresh medium with EELB extract (100 ÎŒg/mL) was added to the test wells. The area of the wound remaining and the rate of migration of cells into the scratched area was examined for 72 hours. The scratched area was imaged at given time points with the help of a Rolera emc2 camera attached to an Olympus CK41 microscope (Olympus, Japan). The percentage cell migration rate and the percentage of wound remaining were calculated using the formulas; percentage migration rate = [(Area of the wound at 0 hour â Area of the wound at time T1)/Area of the wound at 0 hour] X 100, and the percentage of wound remaining = (Area of the wound at time T1/Area of the wound at 0 hour) X 100.\n\n2.2.5 Western blot analysis\n\nThe results of the MTT assay, colony formation, and wound healing assay suggested the negative effect of EELB on CAL-27 cell proliferation, growth, and migratory ability. To evaluate the molecular alterations behind the observed effects, we further analysed the expression levels of the extracellular signal-regulated kinase (ERK1/2), Akt serine/threonine kinase1 (AKT1), cyclin D1 (CCND1), cadherin-1 (CDH1), cadherin-2 (CDH2) and vimentin (VIM). ERK1/2, AKT1 and CCND1 are well-known inducers of proliferation and survival in cancer cells. Loss of CDH1 and overexpression of CDH2 and VIM, which are linked to the epithelial-mesenchymal transition (EMT), are the frequent events during metastatic spread. The effect of EELB on these proteins would hint at the anticancer properties of L. barbarum against OSCC. The cells treated with DMSO (0.1%) were used as vehicle control and the cell cultured in complete medium without EELB extract served as a control. The western blot analysis was performed based on the previously described as follows. In brief, the proteins were extracted using RIPA buffer supplemented with protease inhibitor cocktail (Sigma Aldrich, USA), and the concentration of proteins was assessed using the Bradford assay kit (Sigma Aldrich, USA). Around 30 ÎŒg of the total protein was separated using 10% SDS PAGE, and transferred onto a Nitran membrane (BioRad, USA). The blots were blocked using 5% bovine serum albumin (BSA) (Himedia, India) for 1 hour and then probed separately with the primary antibodies [p-ERK1/2 (RRID:AB_2315112), ERK1/2 (RRID: AB_390779), p-AKT1 (RRID: AB_329825), AKT1 (RRID:AB_329827), CDH1 (RRID: AB_2291471), CCND1 (RRID: AB_2259616), CDH2 (RRID: AB_2687616), VIM (RRID: AB_10695459) and β-Actin (RRID: AB_330288); 1:5000, Cell Signalling Technology, USA)] overnight at 4°C. The phosphorylation site of AKT1 and ERK1/2 was Ser473 and Thr204/Tyr202, respectively. Following incubation with horseradish peroxidase (HRP) tagged secondary antibodies (1:5000; RRID: AB_2099233, Cell Signaling Technology, USA), blots were washed thrice (10mins each) with tris buffer saline containing 0.01% tween 20 (TBST) and visualized using enhanced chemiluminescence reagent (BioRad, USA). The images were taken using the ImageQuant LAS 4000 (GE Healthcare, USA). The antibody against β-Actin was used as a loading control. The band densities were quantified using ImageJ tool (RRID:SCR_003070, https://imagej.nih.gov/ij/).35\n\n2.2.6 Statistical analysis\n\nAll of the assays were conducted in duplicates and were repeated three times for reliability. The data in the bar graphs represent mean±SD. A p-value less than 0.05 was considered statistically significant.\n\n\n3. Results\n\nThe effect of EELB on the cell viability and colony-forming ability of cancerous cells was analyzed by MTT assay and anchorage-dependent colony formation assay, respectively. At 100 ÎŒg, EELB inhibited cell proliferation with 65.5% cell viability compared to 91.7% viability seen with the vehicle control group (Figure 2A). EELB at 100 ÎŒg/mL significantly inhibited colony growth in CAL-27 cells (Figure 1B,1C). The number of colonies reduced significantly for the treated group compared to the vehicle control group. L. barbarum effectively inhibited colony formation in CAL-27 cancer cells. A reduction in the cancer cellsâ vitality was observed when the CAL-27 cells were exposed to EELB.\n\nThe results of the cell migration assay proved that L. barbarum extract inhibited cell migration by decreasing the migration rate in CAL-27 cells. The distance between the edges of the wound was narrow at 24 hours, and it closed completely at 72 hours period in the control group (Figure 3A). Treatment with L. barbarum lowered the migration of CAL-27 cells. At 72 hours, the percentage of the wound remaining for the control and the DMSO group was 11.5% and 14.3%, respectively (Figure 3B). Nevertheless, at the end of 72 hours, L. barbarum extract was successful in inhibiting cell migration of CAL-27 cells. Approximately 42.8% of the wound remained at 72 hours in the treated group. Further, there was a significant reduction in the migration rate upon L. barbarum extract treatment (57.19%) as opposed to control cells (88.47%) (Figure 3C). These results indicate the anti-migratory potential of L. barbarum against CAL-27 cells.\n\nThe cell treated with L. barbarum caused a reduction in the AKT1 and ERK1/2 phosphorylation. The reduction in AKT and ERK levels upon EELB treatment indicates its role in controlling cell proliferation and cell survival. Further, higher CDH1 protein expression along with reduced levels of CDH2, VIM, and CCND1 was noted in the group treated with EELB compared to the control group (Figure 4A, 4B). Increased CDH1 and reduced CDH2 and VIM levels support the beneficial effect of ELLB in inhibiting cell migration and EMT in oral cancer cells.\n\n\n4. Discussion\n\nThe present study assessed for the first time the anticancer potential of goji berry against OSCC. The results showed that goji berries extract can be tried for the treatment of oral cancer. The reduction in cell viability and effective inhibition of colony formation of cancer cells indicate that goji berry extract has antiproliferative properties. Additionally, EELB was found to reduce the migration of cancer cells, which in turn reflects its role in controlling the adhesion of cancer cells to the epithelial surface. EELB also affected the expression of ERK1/2, AKT1, CDH1, CDH2, VIM, and CCND1 proteins. This indicates its role in controlling the growth and migration of oral cell cancers. The anticancer properties of L. barbarum berry can be attributed to the constituents in the berry. L. barbarum berry contains abundant polysaccharides (LBPs), scopoletin, flavonoids, vitamin C analogs, carotenoids (β-carotene and zeaxanthin), β-sitosterol, cerebroside, betaine, amino acids, minerals, and vitamins (in particular, riboflavin and thiamin). Among all these constituents, the major active anticancer compounds in L. barbarum include scopoletin, LBPs, and 2-O-bD-glucopyranosyl-L-ascorbic acid.20â22 These compounds are known for their anticancer, antioxidant, and immunomodulatory properties against important mediators for cell cycles such as ERK, Cyclin D, and AKT.20,27,29\n\nERKs and AKT are some of the most crucial proteins that modulate cancer cell survival and proliferation. AKT helps in metastasis and is associated with the aggressiveness of the tumor.36 AKT activation even inhibits the expression of proapoptotic proteins, such as BAD and BAX, and allows the cancer cells to survive.37,38 AKT can also inactivate the caspase enzymes, which are directly involved in cell apoptosis and forkhead box protein O1 (FOXO-1) expression. This also increases the risk of cell proliferation. AKT can also upregulate cyclin D1, another critical mediator regulating cells to pass the G1 phase of the cell cycle and enter the S phase of the cell cycle. Our results found a reduction in ERK1/2, CCND1, and AKT1 expression, and these findings are important as they indicate a positive effect of EELB in controlling the proliferation and survival of cancer cells.35â38 High expression of CDH1 could also be related to its role in tumor differentiation and inhibition of metastases. High CDH1 levels could increase cellular adhesion in epithelial tissues and reduce the risk of invasion from the epithelium to the connective tissue. A significant decrease in CDH2 and VIM in the group treated with EELB compared to the untreated and vehicle control group also supports the anti-EMT properties of EELB and its role in inhibiting the migration of CAL-27 cells. The regulation of cyclins and cadherins indicates that EELB could prevent the transition from the G1 to the S phase of the cell cycle and EMT.\n\nThese results are similar to previous studies where L. barbarum was found to modulate the levels of cyclins and CDKs such as CDK2, cyclin E, and CCND.25 Activation of EMT signaling helps in cell invasion and migration. Besides, studies have reported that EMT activation can promote the migration of cells. Besides, several studies have reported that EMT inhibition could inhibit cell migration and proliferation.34â38 Previous studies have shown that L. barbarum can inhibit the cell cycle by regulating the expression of p53, p21, and BAX.23 Zhang et al. and Luo et al. found that L. barbarum can break the strands of DNA and induce apoptosis with reduced BCL-2/BAX expression.35,36,38,39 According to Cao et al. (1994), L. barbarum can even activate the macrophages and reduce lipid peroxidation, resulting in the death of cancer cells.36 Additionally, LBPs can affect the natural-killer cells (NKs), which in turn enhance the expression of Interferon-gamma and activate the receptor NKP30 on its surface. This increases the secretion of perforin and may induce the lysis of cancer cells.40â44 Based on our results and the existing evidence supporting the anticancer properties of Lycium barbarum, these results must be validated and confirmed by future clinical and patient-based studies.\n\n\n5. Conclusion\n\nL. barbarum has shown good anticancer properties in in-vitro settings. However, one should further explore its efficacy via clinical trials for treating oral cancer. Further studies can aim to evaluate and compare the role of L. barbarum compared to other anti-cancer agents. Additionally, future research at molecular and genomic levels can be undertaken to evaluate the other molecular mechanisms, signaling pathways, and genes by administrating L. barbarum in patients with OSCC. This evidence could help to develop novel therapeutic strategies for the management of one of the major public health and economic burdens, oral cancer.\n\nThe study was conducted after receiving ethical approval from Kasturba Medical College and Kasturba Hospital Ethic committee with IEC no (460/2019).",
"appendix": "Data Availability\n\nFigshare: [Raw data for effect of goji berry on CAL-27 cell lines (oral squamous cell carcinoma cell line]. https://doi.org/10.6084/m9.figshare.21716309. 45\n\nThe data is also available upon request via email to Dr. Aditi Chopra (email id: aditi.chopra@manipal.edu).\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication)\n\n\nAcknowledgements\n\nWe acknowledge Dr. TMA Pai Structured Ph.D. fellowship program of MAHE, and senior research fellowship from ICMR (Reference ID2019/4115/CMB/BMS), Government of India, for financial assistance to Divya Adiga. All the authors thank Manipal Academy of Higher Education, Manipal for infrastructure support.\n\n\nReferences\n\nWong T, Wiesenfeld D: Oral Cancer. Aust. Dent. J. 2018; 63: S91âS99. Publisher Full Text\n\nMontero PH, Patel SG: Cancer of the oral cavity. Surg. Oncol. Clin. N. Am. 2015; 24(3): 491â508. 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(accessed on 19 December 2021).Reference Source\n\nSchoonees A, Visser J, Musekiwa A, et al.: Pycnogenol® (extract of French maritime pine bark) for the treatment of chronic disorders. Cochrane Database Syst. Rev. 2012; 18(4): CD008294. Publisher Full Text\n\nNewman DJ, Cragg GM: Natural Products as Sources of New Drugs from 1981 to 2014. J. Nat. Prod. 2016; 79(3): 629â661. PubMed Abstract | Publisher Full Text\n\nSeca AML, Pinto DCGA: Plant Secondary Metabolites as Anticancer Agents: Successes in Clinical Trials and Therapeutic Application. Int. J. Mol. Sci. 2018 16; 19(1): 263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLichota A, Gwozdzinski K: Anticancer Activity of Natural Compounds from Plant and Marine Environment. Int. J. Mol. Sci. 2018 9; 19(11): 3533. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTalib WH, Alsayed AR, Barakat M, et al.: Targeting Drug Chemo-Resistance in Cancer Using Natural Products. Biomedicines. 2021; 9(10). PubMed Abstract | Publisher Full Text | Free Full Text\n\nNisar S, Masoodi T, Prabhu KS, et al.: Natural products as chemo-radiation therapy sensitizers in cancers. Biomed. Pharmacother. 2022; 154: 113610. PubMed Abstract | Publisher Full Text\n\nKing N, Balneaves LG, Levin GT, et al.: Surveys of Cancer Patients and Cancer Health Care Providers Regarding Complementary Therapy Use, Communication, and Information Needs. Integr. Cancer Ther. 2015; 14(6): 515â524. PubMed Abstract | Publisher Full Text\n\nZhang Q, Wang F, Jia K, et al.: Natural Product Interventions for Chemotherapy and Radiotherapy-Induced Side Effects. Front. Pharmacol. 2018; 9: 1253. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChoudhari AS, Mandave PC, Deshpande M, et al.: Phytochemicals in Cancer Treatment: From Preclinical Studies to Clinical Practice. Front. Pharmacol. 2020; 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPotterat O: Goji (L. barbarum and L. chinense): Phytochemistry, pharmacology, and safety in the perspective of traditional uses and recent popularity. Planta Med. 2010; 76(1): 7â19. PubMed Abstract | Publisher Full Text\n\nAmagase H, Farnsworth NR: A review of botanical characteristics, phytochemistry, clinical relevance in efficacy and safety of L. barbarum fruit (Goji). Food Res. Int. 2011; 44(7): 1702â1717. Publisher Full Text\n\nJin M, Huang Q, Zhao K, et al.: Biological activities and potential health benefit effects of polysaccharides isolated from L. barbarum L. Int. J. Biol. Macromol. 2013; 54(1): 16â23. PubMed Abstract | Publisher Full Text\n\nWang Z, Liu Y, Sun Y, et al.: Structural characterization of LbGp1 from the fruits of L. barbarum L. Food Chem. 2014; 159: 137â142. PubMed Abstract | Publisher Full Text\n\nYang RF, Zhao C, Chen X, et al.: Different methods extract the chemical properties and bioactivities of Goji (Lycium barbarum) polysaccharides. J. Functional Foods. 2015; 17: 903â909. Publisher Full Text\n\nLuo Q, Li Z, Yan J, et al.: L. barbarum polysaccharides induce apoptosis in human prostate cancer cells and inhibits prostate cancer growth in a xenograft mouse model of human prostate cancer. J. Med. Food. 2009; 12(4): 695â703. PubMed Abstract | Publisher Full Text\n\nMiao Y, Xiao B, Jiang Z, et al.: Growth inhibition and cell-cycle arrest of human gastric cancer cells by L. barbarum polysaccharide. Med. Oncol. 2010; 27(3): 785â790. Publisher Full Text\n\nMao F, Xiao B, Jiang Z, et al.: Anticancer effect of L. barbarum polysaccharides on colon cancer cells involves G0/G1 phase arrest. Medical oncology (Northwood, London, England). 2011; 28(1): 121â126. Publisher Full Text\n\nShen L, Du G: L. barbarum polysaccharide stimulates proliferation of MCF-7 cells by the ERK pathway. Life Sci. 2012; 91(9â10): 353â357. PubMed Abstract | Publisher Full Text\n\nCheng J, Zhou ZW, Sheng HP, et al.: An evidence-based update on the pharmacological activities and possible molecular targets of L. barbarum polysaccharides. Drug Des. Devel. Ther. 2014; 17(9): 33â78. Publisher Full Text\n\nKwok SS, Bu Y, Lo AC, et al.: A Systematic Review of Potential Therapeutic Use of L. barbarum Polysaccharides in Disease. Biomed. Res. Int. 2019; 2019(12): 1â18. Publisher Full Text\n\nTian X, Liang T, Liu Y, et al.: Extraction, Structural Characterization, and Biological Functions of L. barbarum Polysaccharides: A Review. Biomolecules. 2019; 21; 9(9): 389. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaufmann B, Christen P: Recent extraction techniques for natural products: microwave-assisted extraction and pressurised solvent extraction. Phytochem. Anal. 2002; 13(2): 105â113. PubMed Abstract | Publisher Full Text\n\nvan Meerloo J , Kaspers GJ, Cloos J: Cell sensitivity assays: the MTT assay. Methods Mol. Biol. 2011; 731: 237â245. Publisher Full Text\n\nLiang CC, Park AY, Guan JL: In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2007; 2(2): 329â333. PubMed Abstract | Publisher Full Text\n\nFranken NA, Rodermond HM, Stap J, et al.: Clonogenic assay of cells in vitro. Nat. Protoc. 2006; 1(5): 2315â2319. Publisher Full Text\n\nBhat S, Adiga D, Shukla V, et al.: Metastatic suppression by DOC2B is mediated by inhibition of epithelial-mesenchymal transition and induction of senescence. Cell Biol. Toxicol. 2021 Mar 24; 38: 237â258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHong KO, Kim JH, Hong JS, et al.: Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells. J. Exp. Clin. Cancer Res. 2009 26; 28(1): 28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang M, Chen H, Huang J, et al.: Effect of L. barbarum polysaccharide on human hepatoma QGY7703 cells: inhibition of proliferation and induction of apoptosis. Life Sci. 2005;18; 76(18): 2115â2124. PubMed Abstract | Publisher Full Text\n\nCao GW, Yang WG, Du P: Observation of the effects of LAK/IL-2 therapy combining with L. barbarum polysaccharides in the treatment of 75 cancer patients. Zhonghua Zhong Liu Za Zhi. 1994; 16(6): 428â431.\n\nHarsha C, Banik K, Ang HL, et al.: Targeting AKT/mTOR in Oral Cancer: Mechanisms and Advances in Clinical Trials. Int. J. Mol. Sci. 2020; 21(9): 3285. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLim J, Kim JH, Paeng JY, et al.: Prognostic value of activated Akt expression in oral squamous cell carcinoma. J. Clin. Pathol. 2005; 58(11): 1199â1205. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuyan T, Li Q, Yang H, et al.: Protective effect of polysaccharides on simulated microgravity-induced functional inhibition of human NK cells. Carbohydr. Polym. 2014; 101: 819â827. PubMed Abstract | Publisher Full Text\n\nChopra A: Raw data for effect of goji berry on CAL-27 cell lines (oral squamous cell carcinoma cell line). figshare. Figure.2022. Publisher Full Text"
}
|
[
{
"id": "159537",
"date": "30 Jan 2023",
"name": "Swagat Ray",
"expertise": [
"Reviewer Expertise Cell and molecular biology",
"proteomics",
"genomics",
"genome stability."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article discusses a study that aimed to evaluate the anticancer properties of the herb Lycium barbarum (commonly known as Goji berry) against oral squamous cell carcinoma. The study used an ethanolic extract of L. barbarum (EELB) and tested its effects on cancer cell growth, proliferation, and migration in vitro. The study found that EELB impeded the growth, proliferation and migration of oral cancer cells and reduced the phosphorylation of certain signaling proteins associated with cancer cell growth. The study also showed that EELB affected the expression of certain proteins involved in the process of epithelial-mesenchymal transition (EMT), a process that allows cancer cells to become more invasive.\nThe results of the study suggest that Goji berry has good antiproliferative and anti-invasive properties against oral cancer cells. The study suggests that Goji berry could be tried as a potential anticancer agent to manage oral squamous cell carcinoma. However, it should be noted that this study was conducted in-vitro and further research is needed to confirm these findings in-vivo.\nThorough proofreading is also recommended to avoid typos (e.g., 'MTT assay' is written as 'MMT assay' in Figure 1; Figure 4B legends - it should be 'vehicle control' instead of 'vector control').\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9291",
"date": "07 Feb 2023",
"name": "Aditi Chopra",
"role": "Author Response",
"response": "Firstly we would like to thank all the reviewers and the editors for taking the time out to review the paper. We are grateful for your valuable suggestions and comments. Responses to the comments are as follows:Â 1. comment 1: limitation of the study Response: We have added the limitation of the present work at the end of the discussion and stated that the current work is done in-vitro settings and future studies should be done to explore the effect of goji berry in clinical settings. 2. comment 2: Thorough proofreading is also recommended to avoid typos (e.g., 'MTT assay' is written as 'MMT assay' in Figure 1; Figure 4B legends - it should be 'vehicle control' instead of 'vector control'). Response: We apologize for the errors from our end. We have rectified the typo errors in the revised manuscript and in the figure section. Thank You."
}
]
},
{
"id": "159541",
"date": "30 Jan 2023",
"name": "Poornima Rajendran",
"expertise": [
"Reviewer Expertise Periodontology and Implantology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMany anti-oxidants have been tried and tested in the filed of dental medicine. The authors have used Goji berry in the present article. Though the methodology is quite similar to many other materials used so far, the authors have explained why goji berry can be employed to treat oral diseases. Especially their adequate discussion for their test results convinces the reader regarding the potential of the aforementioned agent.\n\nPros of the study:\nWell explained methodology.\n\nDiscussion adequately justifies the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9292",
"date": "07 Feb 2023",
"name": "Aditi Chopra",
"role": "Author Response",
"response": "Firstly we would like to thank the reviewers for spending their valuable time reading our work and understanding the importance of natural alternatives to treat oral disease, especially cancer, especially in current times where many medicine and therapy are becoming less effective or have many side effects. We have revised the manuscript according to the reviewers' comments and corrected the typo errors. Thank you."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1563
|
https://f1000research.com/articles/12-227/v1
|
01 Mar 23
|
{
"type": "Research Article",
"title": "Phytochemicals, proximate composition, minerals and volatile oil analysis of Zanthoxylum acanthopodium DC. fruits",
"authors": [
"Denny Satria",
"Aminah Dalimunthe",
"Dewi Pertiwi",
"Mahatir Muhammad",
"Vera Estefania Kaban",
"Nasri Nasri",
"Syukur Berkat Waruwu",
"Aminah Dalimunthe",
"Dewi Pertiwi",
"Mahatir Muhammad",
"Vera Estefania Kaban",
"Nasri Nasri",
"Syukur Berkat Waruwu"
],
"abstract": "Background: The use of herbal plants is adopted as a traditional medicine because of their minimal side effects. Most plants have bioactive ingredients and nutritional content that can potentially be used as treatments. One plant that has the potential to be a source of modern medicine is Zanthoxylum acanthopodium DC. Historically the use of traditional medicine as a treatment has enjoyed a good sense of trust among the public. The purpose of this study was to perform a qualitative phytochemical screening and proximate analysis of samples of Zanthoxylum acanthopodium DC. Methods: Used in this study were the phytochemical screening test using the thin-layer chromatography method and the proximate analysis using the AOAC method, which included measuring the ash, water, carbohydrate, total fat, protein, and crude fiber content of the samples. Hydrodistillation was used to isolate volatile oil from the sample, which was then identified using gas chromatography-mass spectrometry. The research method used is experimental. Results: From the test, it was found that the phytochemical content of Zanthoxylum acanthopodium DC is alkaloids, flavonoids, tannins, saponins, glycosides, steroids, and triterpenoids. Proximate analysis obtained ash content of 6.19%, water content of 6.35%, carbohydrates of 35.4%, total fat of 2.46%, protein of 16.2%, and crude fiber of 33.4%. Mineral test results prove that Zanthoxylum acanthopodium DC contains Pb (<0.07 mg/Kg), Cd (<0.03 mg/Kg), As (<0.03 mg/Kg), Hg (0.0011 mg/Kg), Mn (43.1 mg/Kg), K (321 mg/Kg), Ca (0.22 mg/Kg), Mg (198 mg/Kg), Fe (52.1 mg/Kg), and Na (23.6 mg/Kg). The highest content of essential oil in Zanthoxylum acanthopodium DC is geranyl acetate, with a concentration of 24.26%. Conclusions: This study concludes that the research findings of Zanthoxylum acanthopodium DC indicate that the phytochemical, mineral, and volatile oil content of the sample is strongly related to its potential to be developed as food and medicine.",
"keywords": [
"Zanthoxylum acanthopodium DC",
"essential oil isolation",
"essential oil content",
"proximate analysis",
"mineral analysis",
"GS-MS",
"secondary metabolites",
"TLC."
],
"content": "Introduction\n\nDue to their potent antioxidant qualities, lack of side effects, and economic viability, plants have been studied for their medicinal properties in the modern scientific community. In terms of effectiveness and absence of side effects, alternative medicine systems can always be trusted.1 According to World Health Organization (WHO) statistics, over 50,000 plant species are used in traditional medicines around the world, and about 21,000 plants are employed in alternative medicine. The entire plant or different parts, such as the leaves, stem, bark, root, flower, tuber, and seed, among others, are used to make the drugs.2 Over 30% of all plant species have been used for medical purposes at some point in their history. When compared to ethnomedicine's use in practice, conventional medication practice in the treatment of diseases and infections has embraced a more scientific and broad measurement, particularly in developing nations.3\n\nPhytochemicals are just one of the several secondary metabolites that give medicinal plants their therapeutic potential. Recent research on phenolic compounds has highlighted several of their potential medical benefits.4 Andaliman's phenolic compounds can help people with diabetes heal burns by making more of a protein called VEGF (vascular endothelial growth factor)5 and are able to inhibit the development of free radicals, so many tests are carried out as an antioxidant.6 The phenolic mixtures' abilities to scavenge free radicals and lessen lipid peroxidation are linked to their anticarcinogenic, antimutagenic, and cardioprotective effects.7\n\nZanthoxylum acanthopodium DC is also used to treat dysentery. Andaliman has been utilized by Indians to treat leprosy and skin conditions like abscesses and paralysis.8 In North Sumatera, notably in North Tapanuli, andaliman has been utilized as a spice.7,8 Numerous substances, including phenol hydroquinones, flavonoids, steroids/triterpenoids, tannins, glycosides, volatile oils, alkaloids, coumarins, lignans, amides, and terpenes, are found in plants belonging to the Zanthoxylum genus.9,10 It has been demonstrated that the andaliman fruit ethylacetate extract (EAF) has cytotoxic effects on the MCF-7 and T47D cell lines. It was discovered that doxorubicin and EAF worked in harmony.11 By a cardioprotective impact and action on T47D resistant cells, EAF was demonstrated to have anticancer activity in mice induced with benzo(a) pyrene.11 Given the aforementioned background, the researchers are interested in performing proximate analyses, secondary metabolite components, and essential oil concentration found in Zanthoxylum acanthopodium DC.\n\n\nMethods\n\nThe material used in this study was the fresh fruit of Zanthoxylum acanthopodium DC. Green-black in color, round in shape, and weighing 5 grams on average, this fruit was collected in Onan Rugu Village, Samosir Regency, North Sumatra Province, Indonesia.\n\nAndaliman fruit is washed with running water and then dried at room temperature. After drying, it was ground using a blender. The sample was blended until smooth, until it looked like powder, to get Zanthoxylum acanthopodium DC powder.9\n\nThe identification of secondary metabolites was carried out using the thin-layer chromatography (TLC) method. A total of 100 mg of extract was dissolved in 1 ml ethanol and then smeared on the stationary phase. The stationary phase was a plate coated with silica gel 60 F254 (Merck, Germany) measuring 10x5 cm. The scale was the inserted into a chamber that has been saturated with mobile phase vapor.12,13 Phytochemical screening tests can be seen in Table 1.\n\nThe hydrodistillation method was used to isolate essential oils. The Zanthoxylum acanthopodium DC powder was placed in a long-necked flat-bottom flask with distilled water and distilled for 4-5 hours. The essential oil was collected in a separatory funnel, and then the essential oil was separated from the water. 1ml of the anhydrous sodium sulfate was added to the essential oil, shaken, let stand for 1 day, and stored in a dark bottle.14\n\nThe determination of essential oil components was carried out using a Gas Chromatograph-Mass Spectrometer (GC-MS; MS). An Rtx-5 MS capillary column (Restek) with a column length of 30 meters, a column diameter of 0.25 mm, an injector temperature of 270 C, and a flow rate of 1.16 ml/minute were used in the analysis. The column temperature was programmed (temperature programming) with an initial temperature of 60 C for 5 minutes, then increased slowly at an increase rate of 5.0 C/minute until the final temperature was 280 C, which was maintained for 30 minutes with the ionizing electron impact (EI) type.\n\nThe way to identify essential oil components is to compare the mass spectra of the essential oil components obtained with the mass spectra in the data library that have the highest similarity index.10\n\nThe AOAC method was used to identify to quantify the amounts of ash, water, carbs, total fat, protein, and crude fiber.15 used toy\n\nThe gravimetric method was used to calculate how much water was in the sample. The sample was weighed until the weight stayed the same, which meant that all the water in the sample had evaporated. The procedure for analyzing the water content is that the cup to be used is dried beforehand at a temperature of 100â105°C for 30 minutes. Then it was dried in an oven at 100â150 C for 6 hours and cooled again in a desiccator for 30 minutes.16\n\nThe ashing method was used to determine the ash content. The principle of this analysis is to burn the material or incinerate it at a high temperature (about 350 °C) and weighing the substance that remains after the ashing.17\n\nProtein content was determined using 0.1 g of powder placed in a 100 ml Kjedhal flask. 2 ml of H2SO4 98% was added and 0.9 g selenium as a catalyst, and allowed to digest for 60 minutes, after which a sample was taken. Distillation was then carried out. The distillate obtained was put in an Erlenmeyer flask containing 15 ml of acid solution, borate 4%, and a mixed indicator (2-3 drops of methyl red and methyl blue). The mixed distillate is titrated with a standard solution in the form of 0.02 N HCl, until a light purple color is obtained.18\n\nA total of 5 g of sample was put in a lead filter, then covered with fat-free cotton wool. Lead containing samples were mounted on a soxhlet extraction unit. The weight is filled with sufficient petroleum benzene (30 ml), then installed on the extraction tool. After the extraction is complete (5 hours), the flask is heated in the oven. It is then put in desicator until all of benzene has evaporated.19\n\nAn analysis of the carbohydrate content was carried out using a different method in the calculated proximate analysis. Using the formula = 100% -%(moisture content + ash content + fat content + protein content), so that the carbohydrate content of the material is obtained.20\n\nNaCl and KCl were used as standards for the mineral analysis, which was done using a flame photometer (Model 405 Corning, UK), after which the sample's mineral content was checked. For all other metals in the sample, an atomic absorption spectrophotometer (Pekin-Elmar Model 403, Norwalk, CT, USA) was used. The determination of each mineral was done twice. All compounds are of analytical grade (BDH, London). Limits for metal detection have previously been established using Techtron. The optimal analytical range has a variable coefficient of 0.87 to 2.20% and is between 0.1 and 0.5 absorbance units. All estimated values are provided as a percentage, even though minerals are reported as milligrams per 100 grams.21\n\n\nResults\n\nThe retention factor (Rf) value is the value or size determined based on the position of the stain on the dissolved substance in thin-layer chromatography. The result of the stain produced by the TLC plate is then computed as the Rf.22,23 Table 2 below shows the findings of the phytochemical analysis of Zanthoxylum acanthopodium DC and the resulting Rf values. According to the analyzed metabolites, stains with different colors were produced by identifying phytochemical substances\n\nAlkaloid compound identification by the TLC method employing chloroform's mobile phase\n\nAfter spraying with dragendroff, methanol: ammonia (85:15:1) appears as a green stain and yields three stains with Rf values of 0.12, 0.2, and 0.72. The utilized mobile phase has a unique polarity. As a result of chloroform having a higher ratio than methanol and ammonia, the mobile phase is more likely to be non-polar. While substances with higher Rf values are more disseminated in the mobile phase, those with lower Rf values are more distributed in the stationary phase. Because they are more strongly held in the stationary phase compared to compounds with higher Rf values, compounds with lower Rf values are more polar and have more significant distribution coefficients.24,25\n\nUsing ethyl acetate to identify flavonoid compounds\n\nOne yellow-orange stain with an Rf value of 0.506 was formed by the mobile phase of ethyl acetate: methanol: water (100:13,5:10). Flavonoids are chemicals that are soluble in polar solvents, and when they are separated using mobile phases with semi-polar and polar solubility qualities, the presence of flavonoid molecules in the sample is increasingly evident26\n\nIdentification of saponin compounds by TLC using the mobile phase Chloroform: acetic acid; methanol: water (11:6:2:1). The results showed 2 brownish yellow spots with Rf values of 0.41 and 0.49 after being sprayed with methanol: sulfuric acid reagent: vanilla.12\n\nIdentification of tannin compounds using the mobile phase of Chloroform-Ethylacetate-n-butanol-Water (11:6:2:1) and 10% FeCl3 stains gave the results of 2 blackish green spots with Rf values of 0.74 and 0.9. This proves that the mobile phase used has the same polarity as tannin, so tannin can be used as an eluent to separate tannin compounds27,28\n\nIdentifying glycoside compounds using the TLC method depends on the mobile phase of ethyl acetate: methanol: water (16: 2: 2) produces 2 stains with Rf 0.2 and 0.62 after spraying with 50% sulfuric acid, giving a brown color.12\n\nIdentification of steroid/triterpenoid compounds by TLC using the mobile phase n-hexane: ethylacetate (8:2) resulted in 2 stains with a purplish-red color, confirming the presence of steroid/triterpenoid compounds.29\n\nThe results of the analysis of essential oils obtained from the gas chromatographyâmass spectrometry (GS-MS) chromatogram contained 57 peaks, and six main components were taken based on the highest concentration. The chromatogram results can be seen in Figure 1. The results of the GS-MS analysis showed six main components, namely: geranyl acetate (24.26%, C12H20O2, m/z 196.29), 1-limonene (20.79%, C10H16, m/z 136.24), citronellol (9.72%, C10H20), m/z 156.27), geraniol (7.32%, C10H18O, m/z 154.25), 1,3,6-octatriene (2.67%, C8H12, m/z 108.18), and beta-ocimene (2.92%, C10H16, m/z 136.23). The physiology of the entire plant is so intertwined with essential oil products that it is reliant on metabolic conditions and established developmental differentiation programs of the produced tissues.30 The essential oil content contained in Zanthoxylum acanthopodium DC can be seen in Table 3.\n\nMeasurements of the ash content, water content, carbs, total fat, and protein were made using proximate analysis. Proximate measures are used to determine the nutritional value of a food product, food ingredient, or food. Analyzing the nutritional value of Zanthoxylum acanthopodium DC will help determine whether it has the potential to be used as a food additive and a therapy. Table 4 displays the findings of the close examination of Zanthoxylum acanthopodium DC. Substance impacts the texture and nutritional formation of the food generated. Bacteria, molds, and yeast can easily thrive in high water content environments and produce changes in the sample's physical or chemical composition.\n\nThe results of the mineral analysis test contained in the Zanthoxylum acanthopodium DC sample can be seen in Table 5 below. The table shows that the mineral content in the Zanthoxylum acanthopodium DC sample is Lead (Pb), Cadmium (Cd), Arsenic (As), Mercury (Hg), Manganese (Mn), Potassium (K), Calcium (Ca), Magnesium (Mg), Iron (Fe) and Sodium (Na).\n\n\nDiscussion\n\nThe fact that Zanthoxylum acanthopodium DC was found to contain phytochemicals such alkaloids, tannins, steroids/triterpenoids, flavonoids, saponins, and glycosides supports its usage as a medicinal plant. These secondary plant metabolites' therapeutic capabilities have also been described by a number of writers.31â34 There is evidence that alkaloids can stimulate the central nervous system.35 The antiviral, antifungal, antibacterial, and anticancer effects of tannins are well documented. They offer a variety of pharmacological qualities that have been linked to wound healing, including anti-inflammatory, analgesic, and antioxidant activities.34,36 The antibacterial properties of tannins are applied through breakdown of membranes, protein binding, adhesion, and inhibition of enzymes.37 According to reports, detected steroids and triterpenoids exhibit anti-bacterial, antiviral, antimalarial, and anti-cholesterol synthesis inhibiting activities.35â37 Antibacterial activities of steroids and triterpenoids have been documented.38,39 They have long been used medicinally, and one of its common biologic characteristics is cytotoxicity.40 Flavonoid content in Zanthoxylum acanthopodium DC has the potential as an antioxidant. Flavonoid can act as antioxidants with the mechanism of chelating or scavenging.41,42 Saponins also detected were known to have the properties of precipitating and coagulating red blood cells.41 Some characteristics of saponins include foam formation in aqueous solutions, hemolytic activity, cholesterol-binding properties and bitterness.43,44 Sodipo et al. (2000) reported that saponins lower cholesterol levels, act as immune boosters and are anti-carcinogenic.43 However, it was also reported that high levels of saponins could cause gastroenteritis.45 There is a low amount of saponins in Zanthoxylum acanthopodium DC, which confirms that its consumption will not harm the human body. In addition, according to many reports, Zanthoxylum acanthopodium DC fruits contain glycosides known to lower blood pressure.46\n\nIn addition to the metabolite content, the results of this study also showed that Zanthoxylum acanthopodium DC contains essential oils. Essential oils are part of a group of volatile compounds and give off a characteristic odor. The most significant ingredients contained in Zanthoxylum acanthopodium DC are essential oils of geranyl acetate (24.26%) and 1-limonene (20.79%). The compounds -myrcene, (z)-β-ocimene, linalool, -citronellol, neral, geraniol, geranyl acetate and sesquiterpenoids contribute to the distinctive aroma of Zanthoxylum acanthopodium DC. Zanthoxylum acanthopodium DC essential oil has potential in vitro cytotoxic effects on T47D breast cancer cells.9,10 Essential oil compounds in Zanthoxylum acanthopodium DC can also inhibit the growth of bacteria.47\n\nThe results of the proximate analysis also show that Zanthoxylum acanthopodium DC has a sample moisture content (of 6.35%). The water content value of this sample still meets the Standard National Indonesia (SNI) No 01-2891-1992 criteria even though it is lower than the range of 15-24% allowed by SNI.48,49 The ash content of the sample is 6.19%, a measure used to determine the number of minerals present in the sample.50,51 Ash content is a metric that indicates the purity of a sample and is affected by its composition and mineral content. The total fat value is 2.46%, the protein value is 16.2%, and the carbohydrate value is 35.4%. A good source of protein was defined as having a protein content that exceeded 12% of its caloric value, and a crude fiber content of 33.4% sample was high enough to meet the recommended daily allowance of fiber.52,53 Carbohydrates and crude fiber, such as lignin and pectin, are the two substances in the samples of Zanthoxylum acanthopodium DC in the highest concentrations. Total fat is the least abundant element in Zanthoxylum acanthopodium DC.\n\nEvery herbal plant provides health benefits because it has nutritional value. Zanthoxylum acanthopodium DC is detected to contain minerals such as Lead (Pb), Cadmium (Cd), Arsenic (As), Mercury (Hg), Manganese (Mn), Potassium (K), Calcium (Ca), Magnesium (Mg), Iron (Fe) and Sodium (Na). Minerals are essential for the proper functioning of tissues and act as secondary messengers in several biochemical cascade mechanisms.54,55 For example, calcium is an important mineral content found in plants because of its many roles in the life of living things. The content of magnesium plays a role in the activity of manganese (Mn), which is considered an antioxidant nutrient and breaks down bile, thereby reducing the risk of cholesterol. Lack of calcium in the body can cause several disorders, for example, inhibiting growth.1,10,53 Potassium in Zanthoxylum acanthopodium DC plays an essential role in diuretics and regulates cardiac function, water, and ion balance in blood and tissues.56,57 Calcium content is well known for the growth and maintenance of bones, teeth and muscles.58,59 In addition, calcium is helpful in the formation of blood and intracellular and extracellular fluids inside and outside the body's cells.60 The iron content is also essential. Iron plays a vital role in erythropoiesis and many intracellular oxygen transport reactions. It facilitates the oxidation of carbohydrates, proteins and fats.61â63 Sodium and potassium are needed to maintain the osmotic balance of body fluids and body pH, regulate muscle and nerve irritability and control glucose uptake.63\n\n\nConclusions\n\nThe study's findings by Zanthoxylum acanthopodium DC demonstrated that the sample's phytochemical, mineral, and essential oil content is strongly associated with its potential to be developed as food and medicine. Every plant must include the biological substances required to serve as metabolites for creating novel medications and foods, so that more comprehensive studies on the effectiveness and compound interactions of these compounds' contents might be developed.",
"appendix": "Data availability\n\nZenodo: The dataset of âPhytochemicals, Proximate Composition, Minerals and Volatile Oil Analysis of Zanthoxylum acanthopodium DC. Fruitsâ. https://doi.org/10.5281/zenodo.7355506. 64\n\nThis project contains the following underlying data:\n\n⢠Table 1. Qualitative Phytochemicals analysis with TLC.xlsx\n\n⢠Table 2. Zanthoxylum acanthopodium DC secondary metabolite screening results.xlsx\n\n⢠Table 3. Volatile oil data of Zanthoxylum acanthopodium DC.xlsx\n\n⢠Table 4. Essential oil content in Zanthoxylum acanthopodium DC.xlsx\n\n⢠Table 5. Proximate analysis data of Zanthoxylum acanthopodium DC.xlsx\n\nZenodo: Picture of TLC result of Zanthoxylum acanthopodium DC secondary metabolite and Chromatogram of essential oil of Zanthoxylum acanthopodium DC. https://doi.org/10.5281/zenodo.7355669. 65\n\nThis project contains the following underlying data:\n\n⢠Figure 1. Picture of TLC result of Zanthoxylum acanthopodium DC secondary metabolite.png\n\n⢠Figure 2. Chromatogram of essential oil of Zanthoxylum acanthopodium DC.jpg\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nNaqbi KMAA, Karthishwaran K, Kurup SS, et al.: Phytochemicals, Proximate Composition, Mineral Analysis and in vitro Antioxidant Activity of Calligonum crinitum Boiss. Horticulturae. 2022; 8(2): 156. Publisher Full Text\n\nSikdar M, Dutta U: Traditional phytotherapy among the Nath people of Assam. Studies on Ethno-Medicine. 2008; 2(1): 39â45. Publisher Full Text\n\nFarnsworth NR: Ciba Foundation Symposium 185-Ethnobotany and the Search for New Drugs. 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Sci. 2018; 22(2): 5.\n\nDashti A, et al.: Estimation of biomass higher heating value (HHV) based on the proximate analysis: Smart modeling and correlation. Fuel. 2019; 257: 115931. Publisher Full Text\n\nLiu K: Effects of sample size, dry ashing temperature and duration on determination of ash content in algae and other biomass. Algal Res. 2019; 40: 101486. Publisher Full Text\n\nGiyatmi G, Zakiyah D, Hamidatun H: KARAKTERISTIK MUTU PUDING PADA BERBAGAI PERBANDINGAN TEPUNG AGAR-AGAR DAN JUS OKRA. Jurnal Teknologi Pangan dan Kesehatan (Journal of Food Technology and Health). 2022; 4(1): 11â19. Publisher Full Text\n\nAja PM, et al.: Comparative proximate and mineral composition of Moringa oleifera leaf and seed.2013.\n\nAdegbe AA, Larayetan RA, Omojuwa TJ: Proximate analysis, physicochemical properties and chemical constituents characterization of Moringa oleifera (Moringaceae) seed oil using GC-MS analysis. Am. J. Chem. 2016; 6(2): 23â28.\n\nGarten CT Jr: Multivariate perspectives on the ecology of plantmineral element composition. Am. Nat. 1978; 112(985): 533â544. Publisher Full Text\n\nAntia BS, Akpan EJ, Okon PA, et al.: Nutritive and anti-nutritive evaluation of sweet potatoes (Ipomoea batatas) leaves. Pak. J. Nutr. 2006; 5: 166â168. Publisher Full Text\n\nMclvor ME, Cummings CE, Mower MM, et al.: Sudden cardiac death from acute fluoride intoxication. The role of potassium. Ann. Emerg. Med. 1987;1894â1897.\n\nNational Research Council (NRC): Recommended Dietary Allowances. Washington DC: National AcademyPress; 1989.\n\nDosumu MI: Chemical composition of the fruit of Tetraptera and the physiochemical properties of its oil. Global J. Pure Appl. Sci. 1997; 3: 61â67.\n\nTuran M, Kordis S, Zeyin H, et al.: Macro and micro minerals content in some wild edible leaves consumes in eastern Anatolia. Tailors and Francis; 2003; 129â130.\n\nOlayiwola IO, Abubakar HN, Adebayo GB, et al.: Study of sweet potato (Ipomea batatas Lam) food for indigenous consumption through chemical and nutritive analysis in Kwara State, Nigeria. Pak. J. Nutr. 2009; 8(12): 1894â1897. Publisher Full Text\n\nNwanjo HU: Studies on the effect of aqueous extract of Phyllanthus niruri leaf on plasma glucose level and some hepatospecific marker in diabetic Wistar rats. The Internet Journal of Laboratory Medicine. 2007; 2(2): 55â62.\n\nde Oliverira AC , Perez AC, Merino G, et al.: Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise. Comp. Biochem. Physiol. 2001; 130c: 369â377.\n\nFreitas AM, Schor N, Boim MA: The effect of Phyllanthus niruri on urinary inhibitor of calcium oxalate Crystallization and other factors associated with renal stone formation BJU int.2002; 89(9): 829â834.\n\nDalimunthe A, Pertiwi D, Muhammad M, et al.: The data sheet of \"Phytochemicals, Proximate Composition, Minerals and Volatile Oil Analysis of Zanthoxylum acanthopodium DC. Fruits\" (Version 1). [Dataset]. Zenodo. 2022. Publisher Full Text\n\nDalimunthe A, Pertiwi D, Muhammad M, et al.: Phytochemicals, Proximate Composition, Minerals and Volatile Oil Analysis of Zanthoxylum acanthopodium DC. Fruits. Zenodo. 2022. Publisher Full Text"
}
|
[
{
"id": "216956",
"date": "02 Nov 2023",
"name": "Fiaz Alam",
"expertise": [
"Reviewer Expertise Pharmacognosy",
"pharmacy",
"natural products",
"Quality control of herbal drugs"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for inviting me to review the manuscript âPhytochemicals, proximate composition, minerals and volatile oil analysis of Zanthoxylum acanthopodium DC. Fruitsâ\nThe idea of the manuscript is fine but its not suitable to be indexed in the current form for international readers. The manuscript needs complete overhauling for English grammar and language. The sentences are written very poorly. The arrangement of the paragraphs is not up to the mark. Here are few examples given below, just for guidance, to improve the manuscript:\nRewrite the background. Give background about Z. acanthopodium traditional use.\n\nAuthor starts the method section in abstract with âUsedâ. Please write as âThe method used...â\n\nPlease proofread and rewrite the whole abstract again.\nKeywords: \"Zanthoxylum acanthopodium DC, essential oil isolation, essential oil content, proximate analysis, mineral analysis, GS-MS, secondary metabolites, TLC\" - Donât repeat words in keywords like essential oil. GSMS? Rewrite it.\nNo voucher specimen of plant is mentioned.\nMethodologies are written incomplete like the section âlipid content.â\nWrite in methodologies under âproximate analysisâ how you measured the contents. Like proteins, lipids, carbohydrates etc.\nWhat is GSMS?\nWhat is Rate% in table 3?\nDecision:Â Major revision required.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-227
|
https://f1000research.com/articles/12-226/v1
|
01 Mar 23
|
{
"type": "Clinical Practice Article",
"title": "Oncoplastic breast volume replacement with the use of lateral intercostal artery perforator flap",
"authors": [
"Sinta Chaira Maulanisa",
"I Gusti Ngurah Gunawan Wibisana",
"I Gusti Ngurah Gunawan Wibisana"
],
"abstract": "Breast cancer is very common, and there has been a lot of progress in terms of diagnosing and treating it. Surgical intervention, which is tailored to the patient's particular needs and numerous clinical concerns, is one of the management methods used. Some indicators that can be used are, for instance, the size and the stage. Smaller and earlier stage malignancies can be treated with a wide excision. However, getting a proper excision margin around the tumor and not removing too much tissue is problematic. Several studies have shown that when 20% of the breast volume is removed, there is a significant risk of deformity. Currently, oncoplastic surgery is the gold standard for breast reconstruction to minimize such a risk, and one of the techniques is volume replacement, which includes reconstruction of the breast with the transposition of tissue from elsewhere. Numerous volume replacement procedures were performed in the reported patients. The thoracodorsal artery perforator (TDAP) and lateral intercostal artery perforator (LICAP) flaps showed good outcomes in terms of cosmetic and oncological aspects, yet LICAP was found to be an excellent choice for the lateral breast defect and the aesthetic result was satisfactory. We present the case of a 19-year-old woman who had a borderline phyllodes tumor and 49-year-old woman with invasive breast cancer ductal carcinoma in situ (DCIS) that was treated with a wide excision and volume replacement with a LICAP. Following the success of the LICAP treatment, the defect appears symmetrical with the contralateral breast a few weeks following surgery. In conclusion, LICAP and TDAP flaps oncoplastic surgery offer advantages that should be considered to increase satisfactory of the patients after breast construction surgery in Indonesia.",
"keywords": [
"Oncoplastic",
"Breast Volume Replacement",
"Lateral Intercostal Artery Perforator Flap",
"LICAP"
],
"content": "Introduction\n\nBreast malignancy has a high incidence and is on the rise all over the world, particularly among women, yet survival rates in underdeveloped countries are lower than in industrialized countries.1,2 The advancement of diagnostic and therapeutic technology has also contributed to the rise in prevalence.3,4 The treatment options for people with breast cancer are extremely varied and tailored to the patient's preferences, particularly for patients with early-stage cancer who are candidates for breast surgery with breast conserving surgery. Both breast conserving surgery (BCS) and mastectomy have an equal chance of survival.5 When surgery is combined with other therapy, BCS is often performed first since it has a better prognosis than mastectomy and one of the methods used is wide excision.6\n\nThe major goal of a wide excision in the breast is to remove the tumor completely while leaving clear surgical margins. When a tumor is minor, a wide excision is used to preserve the breast's natural contour. However, getting a proper excision margin around the tumor and not removing too much tissue, which may result in breast deformity, are at odds. In fact, up to 40% of all patients experience cosmetic failure after a comprehensive breast resection.7 Several studies have demonstrated that once 20% of the breast volume is removed, there is a significant risk of deformity and that the cosmetic consequences of extensive breast excision can be worse than mastectomy in some patients.8 Oncoplastic surgery has evolved as a new surgical method to address this issue, and it is now regarded as the gold standard in breast reconstruction in chosen patients.8 Oncoplastic breast surgery was attempted by Audretsch et al. in 1998. It is the expanded concept of BCS and includes consideration of breast cancer and aesthetics. Two techniques are currently used in oncoplastic surgery according to the excised volume of the breast: the volume displacement technique, which includes using the remaining tissue of the breast to fill the defect, and the volume replacement technique, which includes reconstruction of the breast with the transposition of tissue from elsewhere.9,10\n\nIn oncoplastic surgery, breast size and excised volume are essential factors to consider. The remaining tissue in patients with moderately large breasts is adequate to achieve satisfactory cosmetic results utilizing the volume displacement procedure. After the excision of a small-sized defect, the volume displacement procedure can be used on patients with relatively tiny breasts. If the deformity is moderate or big, however, volume replacement techniques are the only way to provide satisfactory cosmetic results. The tumor's location should also be considered when choosing an oncoplastic volume replacement approach. A perforator flap, such as a lateral intercostal artery perforator (LICAP) flap, a thoracodorsal artery perforator (TDAP) flap, a latissimus dorsi (LD) myocutaneous flap, a lateral thoracodorsal flap, and a thoracoepigastric flap, are examples of volume replacement techniques that use autologous tissue.9\n\nThe LICAP flap has recently acquired popularity, particularly for partial breast reconstruction following lumpectomy or partial mastectomy for tumors in the lateral quadrant. At three points along the intercostal artery, perforators are released. The posterior perforator, located in the lumbar area, the lateral perforator, located in the midaxillary line, and the anterior perforator, located in the anterior chest line, are the three perforators. The anterior border of the latissimus dorsi muscle generally corresponds to the place where the LICAP emerges at the midaxillary line. The perforator continues anteriorly and superficially from here.11 The LICAP flap uses a pedicled perforator flap to fill the defect created by the lumpectomy.10 In this paper, we describe a case series of partial breast reconstruction using lateral intercostal artery perforator in a patient with borderline phyllodes tumor and a patient with invasive breast carcinoma non-specific type grade II, DCIS.\n\n\nCase report\n\nDue to a lump in the right breast in the upper lateral quadrant, a 19-year-old Javenese woman was examined for further assessment. She is an undergraduate student with unremarkable medical and psycho-social history, including her familyâs. According to local inspection, the tumor was 35 mm in diameter, a well-circumscribed oval tumor, movable with respect to the skin and chest wall, and frequently uncomfortable. She had breast ultrasonography, which revealed a well-defined hypoechoic lesion in the right breast in the upper lateral quadrant, 4 cm from the papilla. There was no evidence of increased vascularity or intralesional calcification. The mass on ultrasonography appeared to be benign. The patient underwent excisional biopsy of the right breast in a private hospital. A borderline phyllodes tumor was discovered during the biopsy.\n\nShe was then referred to Dr. Cipto Mangunkusumo Hospital. She had a wide excision and volume replacement with a LICAP flap. During physical examination one month after the first surgery, on inspection there were surgery scar, no lump was found during palpation. However, in a case of phyllodes tumor, a wide excision up to 1 cm outside the margin of the tumor mass was still needed hence, further evaluation was done. Prior to the second operation, she had an ultrasound examination, which revealed an inhomogeneous hypoechoic lesion in the right breast that was irregular in shape and borders, with no enhanced intralesional vascularity, and was 0.6 x 0.5 x 0.5 cm in size. To get a clear margin, the lumpectomy and skin containing the prior scar were excised down to the plane of the pectoralis major muscle, with resection margins greater than 1.0 cm in relation to the neighboring tissues (Figure 1b). The excised tissues were frozen intraoperatively, resulting in a distinct boundary and the discovery of a fibroepithelial lesion.\n\nThe skin substance loss was 10x8 cm in size. After the wide excision, we continued with breast reconstruction to replace the volume that had been lost. At 6th and 7th intercostal region, the LICAP vessels were marked. In a âlazy Sâ toward the lower axilla, a line was drawn along the inferior and lateral mammary folds. The flap was finished with a second âlazy Sâ, drawn inferolaterally (Figure 1a). As with a typical adipocutaneous perforator flap, the flap is lifted over the fascia. The lateral perforators of the intercostal artery were concentrated on the flap that was excised. The LICAP was centered on the perforators found intraoperatively. The first perforator was found to come from the lateral intercostal artery of the 6th intercostal space, while the second perforator was discovered to originate from the lateral intercostal artery of the 5th intercostal space. After tunneling, the LICAP flap raised on both perforators was shifted to the level of substance loss. The drain was introduced once the donor sites were closed. To close the skin defect, the flap was sutured to the defect location (Figure 1c). The defect is slightly symmetrical with the contralateral breast after surgery. During follow-up, five weeks after the surgery, the defect looked naturally symmetrical with a contralateral breast (Figure 2a and 2b).\n\nA 49-year-old woman came to the hospital with complaints of a lump in her left breast in the last 3 months, which was detected during a breast examination screening at the Dr. Cipto Mangunkusumo Hospital. Her ethnicity is Batak and working as a nurse. She had no history of diabetes mellitus type 2, hypertension, genetic abnormalities, or other associated medical conditions. The patient acknowledge that she had no history of smoking and no family members had history of breast lump. She had no prior history of medication or medical interventions regarding her symptoms. Mammography and biopsy were done at the polyclinic, where on the examination it was found to be a marble-size lump. The lump was stable; there was no pain, no orange peel-like appearance, no wounds, or retractions. The mammography showed focal asymmetrical fibroglandula in the left superolateral quadrant of the breast. Ultrasound showed solid lesions at 2-3 o'clock, 2 cm from the left mammary papilla, heterogeneous echogenicity, irregular shape, lobulated borders, parallel orientation, and heterogeneous posterior appearance, with an echogenic rim, measuring about 2.54 x 1.7 x 1.8 cm. On color Doppler, perilesional vascularization was seen.\n\nThere was a hypoechoic lesion at 9 o'clock; 1 cm in the direction of 6 o'clock; and 2 cm from the left mammary papilla; oval shape, well-defined, parallel orientation, without posterior features; size of about 0.5 x 0.3 cm. Color Doppler showed no intralesional or perilesional vasculature. The conclusion of the ultrasound results stated that the left breast mass was categorized as BIRADS 4. The biopsy results found an invasive carcinoma of no special type (NST) oncocytic pattern in breast grade 2, and ductal carcinoma in situ (DCIS) comedo necrosis type core grade 1 was also found in 5% of the tumor area.\n\nIt was decided to have breast conserving surgery (BCS) with wide local excisions and volume replacement using LICAP to restore the defect. Due to the presence of DCIS an intraoperative sentinel lymphnode biopsy (SLNB) with methylene blue and frozen section was done. A lazy S incision was made following the left mammary fold towards craniolateral, a flap was formed towards the tumor, and continued excision of the tumor by encompassing the surrounding normal tissue after injecting methylene blue sub areola at 12 o'clock. The mass was taken and frozen, measuring 8 x 6 x 6 cm. The frozen section revealed a negative superior border at 1.5 cm, a negative lateral border at 2 cm, a negative inferior border at 2.5 cm, a negative medial distance of 2 cm, and a negative tumor base at 1.5 cm distance.\n\nWhen SLNB was done, we extract one lymph node in the intercostobronchial with a negative node from frozen section. Following that, oncoplastic breast reconstruction volume replacement was performed, and the skin paddle was created with LICAP using inframammary and lateral skin. Three perforator arteries have been retained. A drain was implanted after the flap was epithelialized and sutured to fill the defect. The skin paddle was sutured to the skin defect subcuticularly (Figure 3).\n\n\nDiscussion\n\nIn the first case, treatment for phyllodes borderline or malignant is wide excision with a surgical margin of â€1 cm.12 In our situation, an excisional biopsy was performed during the first surgery at the prior facility, with an unclear margin. We presume that the tumor was removed with a tight margin. As a result, some tumor cells are still present. We performed a large excision in the second procedure to obtain a free margin. The tumor was found in the upper lateral quadrant with a defect size of 10x8 cm. In the second case, the patient was confirmed to have invasive breast carcinoma, NST grade II, and DCIS. Surgery was done on the patient using wide excision to confirm lymph node involvement prior to adjuvant therapies. Sentinel lymph node biopsy was done intraoperatively guided with methylene blue. A volume replacement approach in both cases were required due to a substantial decrease in breast volume. The tumor's location should also be considered when choosing an oncoplastic volume replacement approach. To approach the fault, each flap has a favorable arc of rotation. The LICAP flap can be used to restore a lateral breast deformity. This flap has various advantages, including less donor site morbidity, improved flap shaping, improved cosmetic results, and increased patient satisfaction.9\n\nFor breast volume replacement, LICAP flap has many advantages and considered as a good option. However, it also has limitations, such as to harvest the flap and do the breast cancer resection, patient has to be repositioned from lateral to supine position during the surgery. In the other side, the harvesting will leave more apparent breast scar from the lateral mammary fold extends to 5 cm posterior from the posterior axillary line.13,14\n\nTo ensure that the lateral cutaneous branch of the posterior intercostal arteries is included in the flap, the flap's posterior border should be at least 5 cm behind the posterior axillary line. Because perforators from the intercostal arteries emerge at intervals in the lateral chest fold, lateral displacement of the pedicle is feasible. It is preferable to use unidirectional Doppler to find the perforator prior to surgery. The perforator, on the other hand, is found without the use of doppler and is simply marked visually. To examine the perforators and allow easy elevation of the flap, a posterior incision is made first, with an anterior extension at the lower end of the flap. To expose the latissimus dorsi muscle, the incision is made deeper. Between the midaxillary line and the anterior border of the latissimus dorsi muscle, the flap pedicle is located at the level of the inframammary fold. The smaller posterior branch of the lateral cutaneous branch is detected after seeing the anterior border of the latissimus dorsi muscle. The larger anterior branch is found by following this branch.\n\nUsing a volume replacement procedure, individuals with lateral extra tissue who want to keep their present breast form and size may be able to have a wider excision. In our situation, for the first patient, we were able to keep the patient's breast volume and degree of ptosis is similar to the contralateral breast following second surgery. In the first case, however, the flap looked to be larger and darker than the surrounding skin. In the second case, the left breast, which underwent a volume replacement procedure, appeared bigger compared to the contra side because it had a smaller defect than the first patient. Despite such a difference, the aesthetic outcome was not compromised. In some cases, such as partial mastectomy, when the breast volume has been replaced but the ptosis is different from the contralateral side breast, the surgery is commonly paired with mammoplasty procedures to correct the ptosis; nevertheless, this would have required a bilateral procedure for symmetry.13 Two weeks after surgery, the breast appeared more symmetrical, and the conspicuous flap had vanished. The donor site appeared to be in line with the inframammary fold, but with a 5 cm posterior extension incision. The outcomes of this case reveal that abnormalities in all quadrants of the breast can be filled, with the upper lateral quadrant accounting for 95% of the malignancies. The anterior LICAP flap is a safe and effective flap with few problems. Complications were successfully addressed according to national recommendations.\n\nThere are numerous volume replacement procedures reported. Flaps are chosen based on the size of the defect, its location, the surgeon's experience, and local customs. The TDAP flap and the LD flap are two alternative oncoplastic volume surgery options, in addition to the LICAP flap. A fasciocutaneous flap called the TDAP can be used as an alternative. The perforator of the thoracodorsal artery is based on the perforating vessel of the thoracodorsal artery's longitudinal (lateral) terminal branch. These vessels perforate the LD muscle to share in the blood supply of the skin over the back and lateral part of the chest wall.7,12 The LD flap is a myocutaneous flap. It is a composite graft including variable amounts of muscle, skin, and subcutaneous tissue, with a perforator from the thoracodorsal artery. The disadvantage of the LD flap is that we sacrifice the LD muscle.\n\nThe advantage of LD flap is it has enough vascular supply from thoracodorsal artery thus it has less risk of ischemic complication.10,15,16 The disadvantages of harvesting LD muscle were more observed on the donor-site. Regarding the cosmetic and oncological outcome, both TDAP and LICAP flaps have good results. The location and size of the defect, together with the surgeonâs experience may be considered to decide the use of these two flaps.17\n\nDespite the fact that the perforator was short and narrow in our case, LICAP was an excellent choice for the lateral breast defect since the flap could reach the defect without strain and there was no necrosis. The aesthetic result was also satisfactory. The TDAP flap, on the other hand, is more adaptable and can be used in all quadrants. The perforators of TDAP are also short, and they were dissected from the muscle until they contacted the thoracodorsal artery and vein. When a lengthy pedicle and angle rotation are necessary, the perforators should be dissected superiorly until the axillary area. Between the anterior border of the LD muscle and the defect, a subcutaneous tunnel was formed through which the flap was passed before being translated into the breast defect. Extreme caution was exercised at each stage to avoid harming the perforators.17\n\nThe volume displacement procedure can be used following the removal of a small-sized defect in patients with relatively tiny breasts, notably in the Asian population. On the other hand, the volume displacement technique makes it difficult to obtain excellent cosmetic outcomes in patients with moderate or substantial abnormalities after tumor excision. In our scenario, the only way to achieve satisfactory cosmetic results is to use a volume replacement approach with autologous tissue in various types of flaps. Finally, the patient's happiness with the cosmetic outcome was dependent on the right volume replacement approach being chosen in these circumstances, taking into account the removed volume and tumor location.9 To the best of our knowledge, this is the first case report from Indonesia which is reporting the application of TDAP and LICAP flap in Indonesian patients. We are looking forward that TDAP and LICAP could be done more often in cancer centers, considering its satisfactory outcome for the patients. However, the patients were only followed-up for short period and post-operative complications were not observed in this case report.\n\n\nConclusion\n\nSurgery is often an option for patients with breast cancer but getting a proper excision without removing too much tissue is rare. Oncoplastic surgery is now regarded as the gold standard in breast reconstruction in chosen patients. In oncoplastic surgery, breast size and excised volume are essential factors to consider. There are two well-known procedures: volume displacement and volume replacement. Volume displacement procedures can be used on patients with relatively tiny breasts, while volume replacement techniques are the only way to provide satisfactory cosmetic results when the deformity is moderate or large. LICAP, TDAP, and LD are examples of volume replacement techniques that use autologous tissue. TDAP and LICAP flaps showed good outcomes in terms of cosmetic and oncological aspects. Choosing between these two flaps, the decision is influenced not only by the location and size of the defect but may also depend on the experience of the surgeon. LICAP was an excellent choice for the lateral breast defect since the flap could reach the defect without strain and provide a satisfactory aesthetic result. The perforator flap was a suitable reconstructive option with limited donor site morbidity.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patients.",
"appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nSung H, Ferlay J, Siegel RL, et al.: Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021 May; 71(3): 209â249. PubMed Abstract | Publisher Full Text\n\nFrancies FZ, Hull R, Khanyile R, et al.: Breast cancer in low-middle income countries: abnormality in splicing and lack of targeted treatment options. Am. J. Cancer Res. 2020 May 1; 10(5): 1568â1591. PubMed Abstract\n\nBenson JR, Jatoi I: The global breast cancer burden. Future Oncol. 2012; 8(6): 697â702. Publisher Full Text\n\nMahshid G, Neda M, Efat D, et al.: Epidemiology, incidence and mortality of breast cancer in Asia. Asian Pac. J. Cancer Prev. 2016; 17(sup3): 47â52. Publisher Full Text\n\nKeating Nancy L, Edward G, Beth LM, et al.: Treatment decision making in early-stage breast cancer: should surgeons match patientsâ desired level of involvement? J. Clin. Oncol. 2002; 20(6): 1473â1479. PubMed Abstract\n\nde Jana B , Robert S, Johansson Anna LV: Survival after breast conservation vs mastectomy adjusted for comorbidity and socioeconomic status: a Swedish national 6-year follow-up of 48 986 women. JAMA Surg. 2021; 156(7): 628â637. Publisher Full Text\n\nKrekel NM, Zonderhuis BM, Schreurs HW, et al.: Ultrasound-guided breast-sparing surgery to improve cosmetic outcomes and quality of life. A prospective multicentre randomised controlled clinical trial comparing ultrasound-guided surgery to traditional palpation-guided surgery (COBALT trial). BMC Surg. 2011; 11: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoguchi M, Yokoi-Noguchi M, Ohno Y, et al.: Oncoplastic breast conserving surgery: Volume replacement vs. volume displacement. Eur. J. Surg. Oncol. 2016; 42(7): 926â934. PubMed Abstract | Publisher Full Text\n\nJeong L, Min K, Ho P, et al.: Oncoplastic volume replacement techniques according to the excised volume and tumor location in small- to moderate-sized breasts. Gland Surg. 2014; 3: 14â21.\n\nJacobs JED, Al Sanharib S , Ute S, et al.: The anterior LICAP flap: a design option for oncoplastic breast reconstruction. Case rep. plast. surg. hand surg. 2021; 8(1): 158â163. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAcartÃŒrk TO: Lateral intercostal artery perforator-based reverse thoracic flap for antecubital reconstruction. J. Plast. Reconstr. Aesthet. Surg. 2008; 61(11): e5âe8. PubMed Abstract | Publisher Full Text\n\nGradishar WJ, Anderson BO, Jame A, et al.: Breast Cancer, Version 3.2020, NCCN Clinical Practice Guidelines in Oncology. J. Natl. Compr. Cancer Netw. 2020; 18: 452â478. PubMed Abstract | Publisher Full Text\n\nFarid M, Cocco AM, David M, et al.: The Modified Lateral Intercostal Artery Perforator Flap. Plast. Reconstr. Surg. Glob. Open. 2019; 7(2): e2066-e. Publisher Full Text\n\nHamdi M, Van Landuyt K, de Frene B , et al.: The versatility of the inter-costal artery perforator (ICAP) flaps. J. Plast. Reconstr. Aesthet. Surg. 2006; 59(6): 644â652. PubMed Abstract | Publisher Full Text\n\nTarek H, Sherif T, Ahmed O: LICAP Versus TDAP for Reconstruction of Partial Breast Defects.2021.\n\nAbdelrahman Emad M, Nawar AM, Ashraf BM, et al.: Oncoplastic Volume Replacement for Breast Cancer: Latissimus Dorsi Flap versus Thoracodorsal Artery Perforator Flap. Plast. Reconstr. Surg. Glob. Open. 2019; 7: 7. Publisher Full Text\n\nBong KJ, Kyu KD, Woo LJ, et al.: The usefulness of pedicled perforator flap in partial breast reconstruction after breast conserving surgery in Korean women. Arch. Plast. Surg. 2018; 45(1): 29â36."
}
|
[
{
"id": "237802",
"date": "13 Feb 2024",
"name": "Dedy Hermansyah",
"expertise": [
"Reviewer Expertise Surgical oncology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments: The article titled \"Oncoplastic Breast Volume Replacement with the Use of Lateral Intercostal Artery Perforator Flap\" by Sinta Chaira Maulanisa et al., focuses on the use of the lateral intercostal artery perforator (LICAP) flap in oncoplastic breast reconstruction. It presents the cases of two patients, highlighting the effectiveness of LICAP in treating lateral breast defects and providing satisfactory aesthetic outcomes. The study emphasizes the importance of considering oncoplastic surgery options, like LICAP and thoracodorsal artery perforator (TDAP) flaps, for breast reconstruction, particularly in Indonesia. It also addresses the technical aspects and advantages of using LICAP in oncoplastic surgery. To make the article scientifically sound, the authors could consider the following:\nExpanding on comparative studies or data to show the efficacy of LICAP against other reconstructive options. Providing a more detailed analysis of postoperative outcomes and long-term follow-ups to assess the durability of results. Including a larger sample size or a control group to strengthen the validity of the findings. Addressing potential limitations and biases in the study to provide a balanced perspective. Elaborating on the specific criteria for selecting LICAP over other reconstruction techniques, considering patient-specific factors. Discussing potential complications and how they were managed in the cases presented.\nTo provide sharpened criticism for the paper:\nLimited Sample Size: The study's reliance on only two cases significantly limits the generalizability of the findings. For robust conclusions, a larger and more diverse sample is essential. Lack of Comparative Analysis: The paper does not compare LICAP flap outcomes with other reconstruction techniques. Without this comparison, it's challenging to ascertain the superiority or specific indications of LICAP. Insufficient Long-term Data: The study lacks long-term follow-up data, which is crucial to understand the durability and longevity of the reconstructive technique. Methodological Weaknesses: The study design lacks rigor, such as the absence of a control group or randomized trial elements, which are vital for establishing a cause-effect relationship. Under-Addressed Complications and Limitations: The paper does not thoroughly discuss potential complications or limitations of the LICAP flap, which is essential for a balanced view. Patient Selection Criteria Ambiguity: The criteria for choosing patients for LICAP reconstruction are not clearly defined, making it difficult to apply these findings in a broader clinical context. Lack of Statistical Analysis: There appears to be a lack of detailed statistical analysis to support the conclusions, which is a critical aspect of scientific studies.\nAddressing these points would greatly enhance the scientific rigor and credibility of the study.\nTo critically assess the methodology of the paper:\nSample Selection: The study's methodology appears limited by the small number of cases. Using only two patients does not provide a statistically significant sample size, which is crucial for generalizing the results. Lack of Control Group: Without a control group, it's difficult to attribute the outcomes solely to the LICAP flap technique. A comparative study with another reconstruction method would have strengthened the results. Data Collection and Analysis: The methodology lacks detailed information on how data was collected and analyzed. A more rigorous approach, including statistical analysis, is necessary for scientific studies. Follow-up Duration: The follow-up period for the patients is not specified or seems insufficient. Longer follow-up is needed to assess the long-term efficacy and complications of the procedure. Methodological Rigor: The study could benefit from a more structured and detailed methodological approach, outlining the step-by-step process of patient selection, surgery, and postoperative care.\nAddressing these methodological concerns would significantly improve the scientific validity of the study.\n\nIs the background of the casesâ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Yes",
"responses": []
},
{
"id": "257165",
"date": "26 Apr 2024",
"name": "Kanchana Wijesinghe",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn case 1 â the histology of the 1st surgery with the margin clearance should be mentioned. The authours have performed a wide skin resection and replacement. Was this really necessary or could it have been avoided for better aesthetic outcome? The authors can also discuss the option of modified LICAP, which could have been a better option for Case 1.\nIn case 2, it is stated that SLNB was performed for DCIS. But the patient is already diagnosed with invasive cancer. This is contradictory and needs correction.\nLTAP flap is also a good flap to be considered and should be added to the discussion.\n\nIs the background of the casesâ history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-226
|
https://f1000research.com/articles/8-1200/v1
|
26 Jul 19
|
{
"type": "Data Note",
"title": "On the design of linked datasets mapping networks of collaboration in the genomic sequencing of Saccharomyces cerevisiae, Homo sapiens, and Sus scrofa",
"authors": [
"Mark Wong",
"Rhodri Leng",
"Rhodri Leng"
],
"abstract": "This paper describes a unique two-step methodology used to construct six linked bibliometric datasets covering the sequencing of Saccharomyces cerevisiae, Homo sapiens, and Sus scrofa genomes. First, we retrieved all sequence submission data from the European Nucleotide Archive (ENA), including accession numbers associated with each species. Second, we used these accession numbers to construct queries to retrieve peer-reviewed scientific publications that first linked to these sequence lengths in the scientific literature. For each species, this resulted in two associated datasets: 1) A .csv file documenting the PMID of each article describing new sequences, all paper authors, all institutional affiliations of each author, countries of institution, year of first submission to the ENA, and the year of article publication, and 2) A .csv file documenting all institutions submitting to the ENA, number of nucleotides sequenced, number of submissions per institution in a given year, and years of submission to the database. In several upcoming publications, we utilise these datasets to understand how institutional collaboration shaped sequencing efforts, and to systematically identify important institutions and changes in network structures over time. This paper, therefore, should aid researchers who would like to use these data for future analyses by making the methodology that underpins it transparent. Further, by detailing our methodology, researchers may be able to utilise our approach to construct similar datasets in the future.",
"keywords": [
"Bibliometrics",
"Bibliographic Database",
"network analysis",
"genomics",
"S. cerevisiae",
"Homo sapiens",
"Sus scrofa",
"history of science"
],
"content": "Introduction\n\nThis paper describes the methodology used to construct six novel datasets for the European Research Council funded project, Medical Translation in the History of Modern Genomics; a project exploring the history of scientific collaboration around DNA sequencing. The datasets contain information specific to the genomic sequencing of Saccharomyces cerevisiae (bakerâs yeast), Homo sapiens (human), and Sus scrofa (domestic pig), and consist of data related to sequence submissions to public databases and co-authorship relations underpinning the description of those sequences in the scientific literature. As part of this project, we have stored all relevant datasets in the data repository at the University of Edinburgh (see Data availability; Wong et al., 2019).\n\nIn what follows, we first describe a unique two-step methodology that involved:\n\n1. Extracting data on sequence submissions to the European Nucleotide Archive (ENA a public, open access database) via automated routines and Application Programme Interfaces (APIs).\n\n2. Linking particular sequence submissions to peer-reviewed publications that first described these in the literature via API queries, which utilised sequence accession numbers to mine Europe PubMed Central and SCOPUS.\n\nWe then discuss our approach to re-structuring and cleaning these data and offer a description of the content of each dataset. Finally, we reflect on the strengths and weaknesses of these datasets and methods.\n\n\nMaterials and methods\n\nThis project entailed a large and unique data collection exercise of over 13 million records, which were retrieved via 30 million API queries to three different databases. This involved a two-step process. First, we retrieved all sequence submission data from the ENA, including accession numbers associated with particular sequence lengths. Second, we used these accession numbers to construct API queries to retrieve peer-reviewed scientific publications that first discussed and linked to these sequence lengths in the scientific literature.\n\nWe retrieved sequence submission data from the ENA for each of the three species over defined periods â S. cerevisiae (1980â2000), H. sapiens (1985â2005), and S. scrofa (1990â2015). The date ranges for each species were selected based on the history of science objectives underlying our project. The purpose was to capture submissions before, during, and after the completion of concerted efforts to systematically sequence the genome of each of the organisms. The search was conducted by making a series of calls to ENAâs API for each species and each year the study investigated. The query was constructed by specifying the taxonâs number (tax_eq) in the ENA index (i.e. 9606 for H. sapiens, 4932 for S. cerevisiae and 9823 for S. scrofa) and the sequence release date (first_public) to filter records that were released within a certain year. The search parameter of first_public was specified as âgreater than or equal toâ 1st January and âless than or equal toâ 31st December of the year. Additional parameters were used to specify search for sequence release records (result=sequence_release) and download the data in .XML format (display=xml). In cases where records per year exceeded the ENAâs limit of 100,000 records per API call, the pagination function (offset) was deployed.\n\nThis procedure allowed us to mine the ENA database based on the species and years relevant to our study and extracted data on: 1) the number of nucleotides submitted for each of these species; 2) all accession numbers associated with these sequence lengths; 3) the date of submission; 4) the name of the submitting individual and their institutional affiliation (if available); and 5) papers in the scientific literature associated with each accession number (if specified by the submitter) (Li et al., 2015). Further details about the API queries are contained in the R scripts made available together with the datasets (see Software availability; UofGMarkWong, 2019). As the ENA is part of The International Nucleotide Sequence Database Collaboration (INSDC), which facilitates the sharing of information of three main sequence databases, including the European Nucleotide Archive, based in the European Bioinformatics Institute, GenBank, provided by the US National Centre for Biotechnology Information (NCBI), and DNA Data Bank of Japan (DDBJ), we were able to retrieve all sequence submissions from institutions participating in these databases. Once collected, we utilised the R statistical environment (R Development Core Team, 2016) to structure these data for further cleaning and analysis. In Table 1, we report the total records retrieved via this process.\n\nHowever, as the availability of submitter information was found to be sparse and the ENA often lists one submitting institution only (see Table 1), we used publication data as a proxy to identify collaboration between institutions. We generated queries to the Europe PubMed Centralâs (Europe PMC) API by using the ENA accession number as a parameter to search for associated publications (EMBL_PUBS). This linkage allowed us to identify a list of PubMed IDs (PMIDs) of the publications linked to these accession numbers (Lopez et al., 2014). In addition, other parameters were used including result_type=core to return full metadata available, format to download as .JSON files, cursorMark as a pagination option, and the default result limit of each API call (pageSize) at 1000 publication records. We deployed a routine to automate the search for each accession number in our dataset. The routineâs procedure to compose and make an API call to Europe PMC using a list of accession numbers (pre-extracted using the ENA API call detailed) have been made available in an online repository (see Software availability; UofGMarkWong, 2019).\n\nWe then extracted fuller data on all authors, their institutional affiliations, the city and country of institution and the date of publication in SCOPUS using the PMIDs as a search parameter (PMID) and utilising other default parameters such as apikey, apart from view=complete to specify returning of full meta-data. The routines and R scripts used are also available (see Software availability; UofGMarkWong, 2019). The use of two bibliometric databases was considered necessary, as while EuropePMC allowed searches for publications linked to and specifically describing an accession number, it only holds institutional information of the corresponding author for all publications published before 2014 (Europe PMC Consortium, 2015). SCOPUS holds fuller bibliometric records of all authors and their institutions, particularly for biomedical and natural science literature (Rotolo & Leydesdorff, 2015). This was crucial for mapping institutional collaboration. However, this database only allows searches based on its text-mining functions and returns publications that mention an accession number anywhere in the text â thus the necessity of inputting the PMIDs retrieved via EuropePMC.\n\nWe selected only the first articles to be published associated with an accession number because first publications are more likely to be written by the groups responsible for the submission of the original version of the sequence (either to describe their contribution or to use it in agricultural or biomedical research). Although this correspondence between submission and first publication is not universal, our search strategy excluded papers that utilised particular sequence lengths that had already been described in the literature and consequently refined our corpus of PMIDs (see differences between Table 1 and Table 2).\n\nOnce collected, researchers in our team cleaned these datasets via VantagePoint (2017) v.10 by using a combination of fuzzy logic algorithms available in the software (i.e. âFuzzy word matchingâ to make fuzzy word comparisons at 95% or lower) and manual cleaning to standardise institution, author and country names according to a pre-specified protocol. The protocol specified and ensured consistency in name conventions, fully spelling out acronyms and abbreviations, removing articles and legal entities, using proper case conventions, removal of white spaces and ineligible characters, removal of duplicates, and keeping school and department data if it appears more than 50 times in the dataset. Missing data, particularly regarding institutional affiliation, was filled manually by scrutinising the record on SCOPUSâ web front-end. To replicate this cleaning process, other open source software, such as OpenRefine, may also be used as an alternative.\n\nIn total, each species has two associated datasets: 1) A .csv file documenting the PMID of each article describing new sequences, all paper authors, all institutional affiliations of each author, countries of institution, year of first submission to the ENA, and the year of article publication, and 2) A .csv file documenting all institutions submitting to the ENA, number of nucleotides sequenced, number of submissions per institution in a given year, and years of submission to the database.\n\nThe ENA dataset documents the volume of DNA sequencing per year and per institution, as measured by either the number of sequence submissions to the database or the number of nucleotides sequenced (where the submitterâs institution information is known and recorded in the submitter fields in ENA). In the datasets we provide, these records are linked to specific submitting institutions per year.\n\nThe dataset of publications includes all data necessary to construct co-authorship networks of collaboration between individuals, institutions and countries that were involved in these sequencing efforts. Table 2 contains the figures for the total number of unique publications (PMIDs) we hold for each species (where the required information on author institutions could be automatically retrieved from SCOPUS) and the total number of institutions and countries involved in authoring these publications.\n\nOur study reflects that the growing capacity in data infrastructure and the development of bioinformatics offers new opportunities not only for life scientists and molecular biology but also for social scientists and historians of science. The method outlined in this paper provides a novel source of evidence to evaluate the development and growth of collaboration in DNA sequencing and genomics research. It is also able to avoid placing a narrow focus on a number of key players based on previous studies or historical accounts. Our datasets show a diversity of countries and institutions involved in the sequencing of the human, yeast and pig genomes. Thus, they enable us to complement previous historical studies that have been focused on a limited number of large-scale sequencing centres (e.g. Hilgartner, 2017).\n\nThis analysis is, however, limited by the data infrastructure that we have used. Its organisation and, especially, its absences can indeed shape and affect how and what we can know about the past; how and what information is being recorded, what is missing, what can and cannot be automatically retrieved, what is considered important (or not), and for what questions the information was expected to provide answers to. These processes, including storage and curation, were built into the databases and can have significant impacts on what we know and what we can study about collaborations in genomic sequencing. For instance, in the ENA, a proportion of accession numbers did not have any further information about submitters. We need to consider these absences, along with their underlying meanings and power dynamics more carefully, especially when we use digital research methods and online data (Lupton, 2015).\n\nFor this reason, we argue that qualitative work should accompany digital research methods. In our project, we are currently working on a mixed methods approach based on constant, bi-directional interactions between quantitative data and other qualitative historical evidence, such as documents stored in archives. This approach has been especially useful to highlight competing visions and different narratives of genomics, and how these might have changed over time.\n\n\nData availability\n\nEdinburgh DataShare: Human, yeast and pig genomics: sequence submissions and first sequence descriptions in the literature (1980â2015). https://doi.org/10.7488/ds/2589 (Wong et al., 2019).\n\nThis project contains the following underlying data:\n\n- Human_publications.csv (Spreadsheet containing PMIDs and publication information for Homo sapiens sequences)\n\n- human_submissions.csv (Spreadsheet containing institutional and submission information for Homo sapiens ENA submissions)\n\n- Yeast_publications.csv (Spreadsheet containing PMIDs and publication information for S. cerevisiae sequences)\n\n- yeast_submissions.csv (Spreadsheet containing institutional and submission information for S. cerevisiae ENA submissions)\n\n- Pig_Publications.csv (Spreadsheet containing PMIDs and publication information for Sus scrofa sequences)\n\n- pig_submissions.csv (Spreadsheet containing institutional and submission information for Sus scrofa ENA submissions)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\n- Source code available from: https://github.com/UofGMarkWong/TRANSGENE\n\n- Archived source code at time of publication: https://doi.org/10.5281/zenodo.3345686 (UofGMarkWong, 2019)\n\n- License: CC-BY 4.0",
"appendix": "Grant information\n\nThe research reported in this project was funded by the European Research Council Starting Grants scheme [678757].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank the European Bioinformatics Institute, and particularly Web production and the Literature team, for invaluable technical advice and allowing one of the authors to undertake a three-week post-doctoral fellowship at the Wellcome Genome Campus. Our colleagues at University of Edinburgh, Dr. Gil Viry and Dr. Miguel GarcÃa-Sancho, also provided important advice on the methods and their historiographical significance and comments on earlier drafts of this paper. Rodrigo Liscovsky, at the University of Edinburgh, was part of the team cleaning the datasets. The staff at the Edinburgh DataShare repository provided assistance and service in uploading and indexing our datasets.\n\n\nReferences\n\nEurope PMC Consortium: Europe PMC: a full-text literature database for the life sciences and platform for innovation. Nucleic Acids Res. 2015; 43(Database issue): D1042âD1048. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHilgartner S: Reordering Life: Knowledge and Control in the Genomics Revolution. Massachusetts: MIT Press. 2017. Publisher Full Text\n\nLi W, Cowley A, Uludag M, et al.: The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Res. 2015; 43(W1): W580âW584. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLopez R, Cowley A, Li W, et al.: Using EMBL-EBI Services via Web Interface and Programmatically via Web Services. Curr Protoc Bioinformatics. 2014; 48: 3.12.1â50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLupton D: Digital Sociology. Oxon and New York: Routledge. 2015. Reference Source\n\nRotolo D, Leydesdorff L: Matching Medline/PubMed data with Web of Science: A routine in R language. J Assoc Inf Sci Technol. 2015; 66(10): 2155â2159. Publisher Full Text\n\nR Development Core Team: R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. 2016. Reference Source\n\nUofGMarkWong: UofGMarkWong/TRANSGENE: TRANSGENE source codes release. 2019. http://www.doi.org/10.5281/zenodo.3345686\n\nWong M, Leng RI, Viry G, et al.: Human, yeast and pig genomics: sequence submissions and first sequence descriptions in the literature (1980-2015) [dataset]. Science, Technology and Innovation Studies. University of Edinburgh. Stable. 2019. http://www.doi.org/10.7488/ds/2589"
}
|
[
{
"id": "61097",
"date": "03 Jun 2020",
"name": "Rachel Ankeny",
"expertise": [
"Reviewer Expertise History/philosophy of contemporary biological sciences (hence I am not qualified to assess the details of the methodologies utilised in terms of data science but have expertise in use of larger datasets for historical explorations)"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes methodologies used to construct datasets on sequence submissions and co-authorship relationships relating to genomic sequencing of three major organisms. The methodology is clearly described, as are its limitations and prospects for use by other scholars. I particularly appreciated the careful reflections on strengths and weaknesses of the approaches taken, and agree that these approaches have clear prospects for enriching our historical/sociological accounts given tendencies to focus on the strongest (or loudest!) research centres to the neglect of other participants particularly in genomic sequencing efforts. Would strongly suggest citing Leonelli's book1 on data curation (in addition to Lupton's more general work on this topic) as it addresses many of the relevant issues raised in the limitations relating to curation and completeness of data specifically in the context of modern biology.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": [
{
"c_id": "6049",
"date": "21 Oct 2020",
"name": "Mark Wong",
"role": "Author Response",
"response": "Thank you for your positive comments and excellent feedback. We are grateful for your time and appreciate your comments on the originality, significance, and timeliness of this paper. We have adopted your suggestion and cited Leonelli's (2016) seminal work in this area too."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1200
|
https://f1000research.com/articles/11-168/v1
|
11 Feb 22
|
{
"type": "Research Article",
"title": "Effect of Aloe vera extract in post-burn skin repair in rats",
"authors": [
"Lusiana Aulia",
"Yunita Sari Pane",
"Lusiana Aulia"
],
"abstract": "Background: Burn injury is a global health problem that is most often caused by heat. Burn injury can cause high morbidity and mortality and requires high cost. Therefore, the use of plants as herbal medicine has the potential to be developed in Indonesia. Aloe vera contains various active ingredients that help the wound healing process, such as glucomannan and acemannan which have the effects on the proliferation of macrophages, and fibroblasts, and re-epithelialization. This study aimed to determine the effect of Aloe vera extract in repairing post-burn skin in rats that was analyzed from the number of macrophages and fibroblasts, and epidermal thickness. Methods: This is an experimental study with a posttest-only control group design using 54 Rattus norvegicus Wistar strain rats. The sampling method was simple random sampling consisting of 3 groups, i.e., I. standard group, which were normal rats; II. negative control group, which were given second-degree burns and treated with gel base (without Aloe vera extract); III. treatment groups, which were given second-degree burns and treated with Aloe vera extract gel. Each group was subdivided into three smaller groups (n = 6) according to the time the lesions were evaluated. Skin tissue samplings were carried out on day 3, 14, and 21 after injury to observe the number of macrophages and fibroblasts, and epidermal thickness. Results: There were significant differences in the mean number of macrophages, number of fibroblasts, and epidermal thickness in all groups (p<0.05). Conclusion: Aloe vera extract gel could accelerate the healing process of burns in rats.",
"keywords": [
"burn injury",
"Aloe vera",
"macrophages",
"fibroblasts",
"epidermal thickness"
],
"content": "Introduction\n\nBurn injury is a damage to the skin or other tissues that can be caused by heat, radiation, electricity, chemicals, or friction, but most often caused by heat (American Burn Association, 2019). Burn injury that is due to heat consists of three causes, which are burns due to hot liquids (scalds), flames (flame burns), or hot solids (contact burns) (World Health Organization, 2018). Burn injury can be divided into four degrees based on its depth. A first-degree burn is limited to the epidermis. Second-degree burns involve both epidermis and dermis. Third-degree burns involve epidermis, dermis, and dermal appendages. Lastly, fourth-degree burns can penetrate into subdermal fat, underlying fascia, muscle, and/or bone (ABA, 2018).\n\nBurn injury can cause high morbidity and mortality and its treatment requires high cost. This is because of the long-term outpatient care, such as dressing changes and some even require surgical reconstruction (Smolle et al., 2017). WHO stated that around 180,000 deaths every year are caused by burns and majority occur in lower-middle-income countries due to lack of adequate facilities to reduce the incidence of burns (Menkes, 2019; WHO, 2018). According to the data from âRiset Kesehatan Dasarâ RISKESDAS (Basic Health Research, Indonesia) in 2018, the proportion of burn injury in Sumatera Utara was 1% and based on the medical record data from Haji Adam Malik Hospital in Medan, North Sumatra-Indonesia, there were 353 cases of burns in 2011â2014 (Maulana, 2014).\n\nThere are three phases of wound healing, namely the inflammatory, proliferative and remodeling phases. The inflammatory phase is characterized by the migration of leucocytes such as macrophages to burn site to degrade debris or microbes. Next is the proliferative phase where the process of angiogenesis, re-epithelialization, and fibroblast proliferation occurs. Lastly, the remodeling phase is characterized by collagen remodeling, wound contraction, and decreased blood flow (Reinke & Sorg, 2012).\n\nSeveral studies have shown that the use of silver sulfadiazine drug can cause side effects such as leukopenia and renal toxicity (Chaby et al., 2005; Fuller & Engler, 1988). Therefore, alternative treatments such as the use of herbal medicine is relatively safer. A number of medical plants grow in Indonesia due to favorable tropical natural conditions. The information about these herbs is passed down from one generation to another, and they are commonly used in traditional medicine, forming an integral part of Indonesian cultural heritage (Gazali et al., 2013). One of the herbs that can be used for the treatment of burns is Aloe vera. The active chemical constituents found in Aloe vera such as glucomannan and acemannan can stimulate the activity and proliferation of fibroblasts. Acemannan can also increase the number of macrophages and stimulates fibroblast to produce keratinocyte growth factor-1 (KGF-1) for re-epithelialization process (Jettanacheawchankit et al., 2009; Sierra-GarcÃa et al., 2004; Surjushe et al., 2008). This study aimed to determine the effect of Aloe vera extract in repairing post-burn skin in rats that was analyzed from the number of macrophages, and fibroblasts, and epidermal thickness.\n\n\nMethods\n\nThis is an experimental study in an animal burn-model with a posttest-only control group design. This study is reported in lines with the in-vivo Animal Research: Reporting guidelines ARRIVE.\n\nThis study was approved by the Research Ethics Committee of Universitas Sumatera Utara (No. 711/KEP/USU/2021). This study was conducted at the Pharmacology Laboratory of the Faculty of Medicine, Universitas Sumatera Utara.\n\nHealthy male Wistar strain rats, 10â12 weeks old, weighing 150â200 g were purchased from the Department Biology of Mathematics and Scientific Faculty, Universitas Sumatera Utara. Before starting the experiment, the animals were adapted for 7 days. They were kept in plastic cages (30 x 20 x 20 cm) and covered with fine wire mesh; base of cages was covered with rice husks as thick as 0.5â1 cm and replaced every day during the study. They were kept in 12 hours of daylight (06:00 A.M. â 06.00 P.M.) and 12 hours of dark cycle (06:00 P.M. â 06.00 A.M.) and fed with standard feed (CP 551 from PT Charoen Pokphand-Indonesia) and water was given ad libitum. They were kept in cages that were maintained at room temperature and humidity at normal ranges.\n\nAll animals were randomly divided into three big groups by simple random sampling. Group I (standard) consisted of normal rats, group II (negative control) was inflicted a burn wound and treated with gel base, and group III (treatment group) was inflicted with a burn wound and treated with Aloe vera gel. Each group was subdivided into three smaller groups (n = 6 per group) according to the time the when the lesions were evaluated. They were treated twice a day according to the groups.\n\nThe sample size was calculated according to Federerâs formula: (Federer, 1963)\n\nt = the number of groups\n\nnâ=âthe number of samples\n\nnâ= 2.875 â 3 rat/group\n\nConsidering the NC3Rs principal in experimental animal, statistical analysis necessity, and the possibility of drop out, the number of rats per group were determined to be six. All 54 rats were given a number (1â54) using a marker pen, then randomized by putting the numbers in an envelope and dividing them into 9 groups, according to the numbers taken from the envelope (Russell and Burch, 1992).\n\nTo ensure that the rats remain in a comfortable condition, they were given anesthesia before performing the burn. The animals were anesthetized with Ketamine/Xylazine 0.2 mL by intraperitoneal injection (in accordance with the provisions of Vertebrate Animal Research). The hair at dorsal region was shaved and cleansed with alcohol swab. Second-degree burn wound was inflicted using iron plate measuring 2Ã2 cm2 which was warmed in boiling water for 5 minutes and placed for 30 seconds on the shaved area (Kristyaningsih, 2016).\n\nAbout 2000 g of Aloe vera leaves were collected. The bottom part and the serrated edges of Aloe vera leaves were cut and peeled to obtain the inner clear gel. About 1000 g of inner gel was weighed and washed thoroughly and cut it into small pieces. Then, the small pieces of gel and 2000 mL 70% ethanol was blended and poured into a closed container (stirred occasionally for 6 hours, then set aside for 18 hours). This mixture was then filtered to obtain the filtrate (I). The extraction process was repeated on the residue using 1000 mL 70% ethanol to obtain another filtrate (II). The filtrate (I) and (II) were mixed and evaporated using a vacuum rotary evaporator (Heidolph Instruments GmbH & Co. KG, Germany) at 400C to obtain a thick extract.\n\n\n\n⢠Gel base\n\nAbout 400 cc of distilled water was taken into the mortar, then 12 g of 3% Sodium-Carboxymethyl cellulose (Na-CMC) powder was evenly added. It was closed and set it aside for 15 minutes. After which it was ground and 4 g of 1% glycerin was added until a homogeneous gel base was formed.\n\n⢠Aloe vera gel\n\nPut 40 g of Aloe vera extract into the mortar then add 360 g of gel base gradually while grinding it until homogeneous.\n\nSix animals of each groups were sacrificed on the 3rd, 14th, and 21st days after injury by injecting 100 mg/kg ketamine intraperitoneally. Then, the injured tissues were carefully dissected and fixed with 10% formalin. All samples were taken to Anatomic Pathology Laboratory of the Faculty of Medicine, Universitas Sumatera Utara for H&E staining. The number of macrophages and fibroblasts, and epidermal thickness was evaluated using Olympus CX21 light microscope with 400à magnification and three fields of view.\n\nData were analyzed using SPSS version 20 (RRID:SCR_019096) with one-way ANOVA test. If there were significant differences (p < 0.05), then LSD test was conducted to determine the differences among groups. Data were presented as mean ± SD.\n\n\nResults\n\nThe results obtained from the study are shown in Fig. 1â3. From the one-way ANOVA results, there were significant differences in the mean number of macrophages and fibroblasts, and epidermal thickness in all groups (p < 0.05). Then, least Significant Difference (LSD) test was conducted to determine the differences among groups.\n\nI (standard), II (negative control), III (treatment)\n\n*p<0.05 statistically analysis by ANOVA showed significant difference.\n\nI (standard), II (negative control), III (treatment)\n\n*p<0.05 statistically analysis by ANOVA showed significant difference.\n\nI (standard), II (negative control), III (treatment)\n\n*p<0.05 statistically analysis by ANOVA showed significant difference.\n\nOn the 3rd day after injury, the highest mean number of macrophages were seen in the treatment group (III), followed by the negative control group (II), and followed by the standard group (I). From the post hoc test results, it appeared that there were significant differences in the mean number of macrophages (p<0.05) in groups I-3 vs II-3 (0.50 ± 0.45 vs 7.77 ± 0.62; p=0.0001), group I-3 vs III-3 (0.50 ± 0.45 vs 9.00 ± 0.47; p=0.0001), and group II-3 vs III-3 (7.77 ± 0.62 vs 9.00 ± 0.47; p=0.001).\n\nOn the 14th day after injury, the highest mean number of macrophages were seen in the negative control group (II), followed by the treatment group (III), and followed by the standard group (I). From the post hoc test results, it appeared that there were significant differences in the mean number of macrophages (p<0.05) in groups I-14 vs II-14 (0.61 ± 0.39 vs 6.22 ± 0.98; p=0.0001), group I-14 vs. III-14 (0.61 ± 0.39 vs. 5.33 ± 0.42; p=0.0001), and group II-14 vs. III-14 (6.22 ± 0.98 vs 5.33 ± 0.42; p=0.013).\n\nOn day 21st day after injury, the highest mean number of macrophages were seen in the negative control group (II), followed by the treatment group (III), and followed by the standard group (I). From the post hoc test results, it appeared that there were significant differences in the mean number of macrophages (p<0.05) in groups I-21 vs II-21 (0.55 ± 0.40 vs 4.72 ± 0.87; p=0.0001), group I-21 vs III-21 (0.55 ± 0.40 vs 3.16 ± 0.34; p=0.0001), and group II-14 vs III-14 (4.72 ± 0.87 vs 3.16 ± 0.34; p=0.0001).\n\nOn the 3rd day after injury, the highest mean number of fibroblasts were seen in the treatment group (III), followed by the negative control group (II), and followed by the standard group (I). From the post hoc test results, it appeared that there were significant differences in the mean number of fibroblasts (p<0.05) in groups I-3 vs II-3 (3.77 ± 0.34 vs. 4.61 ± 0.49; p=0.047), group I-3 vs III-3 (3.77 ± 0.34 vs. 6.44 ± 0.74; p=0.0001), and group II-3 vs. III-3 (4.61 ± 0.49 vs 6.44 ± 0.74; p=0.0001).\n\nOn the 14th day after injury, the highest mean number of fibroblasts were seen in the treatment group (III), followed by the negative control group (II), and followed by the standard group (I). From the post hoc test results, it appeared that there were significant differences in the mean number of fibroblasts (p<0.05) in groups I-14 vs II-14 (3.94 ± 0.38 vs 8.11 ± 0.68; p=0.0001), group I-14 vs III-14 (3.94 ± 0.38 vs 21.55 ± 1.02; p=0.0001), and group II-14 vs III-14 (8.11 ± 0.68 vs. 21.55 ± 1.02; p=0.0001).\n\nOn the 21st day after injury, the highest mean number of fibroblasts were seen in the negative control group (II), followed by the treatment group (III), and followed by the standard group (I). From the post hoc test results, it appeared that there were significant differences in the mean number of fibroblasts (p<0.05) in groups I-21 vs II-21 (3.72 ± 0.38 vs. 9.05 ± 0.80; p=0.0001), group I-21 vs III-21 (3.72 ± 0.38 vs 6.66 ± 1.05; p=0.0001), and group II-21 vs III-21 (9.05 ± 0.80 vs 6.66 ± 1.05; p=0.0001).\n\nOn the 3rd day after injury, the mean epidermal thickness was highest in the standard group (I), followed by the treatment group (III), and followed by the negative control group (II). From the post hoc test results, it appeared that there were significant differences in the mean epidermal thickness (p<0.05) in groups I-3 vs II-3 (5.88 ± 0.76 vs 2.43 ± 0.45; p=0.0001), group II-3 vs III-3 (2.43 ± 0.45 vs. 5.15 ± 0.36; p=0.0001), while group I-3 vs. III-3 (5.88 ± 0.76 vs. 5.15 ± 0.36; p=0.182) did not show a significant result.\n\nOn the 14th day after injury, the mean epidermal thickness was highest in the treatment group (III), followed by the negative control group (II), and followed by the standard group (I). From the post hoc test results, it appeared that there were significant differences in the mean epidermal thickness (p<0.05) in groups I-14 vs II-14 (6.01 ± 0.54 vs 11.46 ± 0.92; p=0.0001), group I-14 vs III-14 (6.01 ± 0.54 vs 16.75 ± 1.31; p=0.0001), and group II-14 vs III-14 (11.46 ± 0.92 vs. 16.75 ± 1.31; p=0.0001).\n\nOn the 21st day after injury, the mean epidermal thickness was highest in the treatment group (III), followed by the negative control group (II), and followed by the standard group (I). From the post hoc test results, it appeared that there were significant differences in the mean epidermal thickness (p<0.05) in groups I-21 vs II-21 (5.92 ± 0.68 vs. 23.10 ± 1.37; p=0.0001), group I-21 vs III-21 (5.92 ± 0.68 vs 30.47 ± 1.25; p=0.0001), and group II-21 vs III-21 (23.10 ± 1.37 vs. 30.47 ± 1.25; p=0.0001).\n\n\nDiscussion\n\nMacrophages are cells that multiply when there is a wound to phagocytose debris and bacteria and they are the predominant cells on the 3rd day post wound infliction (Gurtner & Wong, 2013). This study showed that both negative control and treatment groups had more number of macrophages than standard group on the 3rd day after injury. On the same day, the treatment group had more macrophages than negative control group because there is acemannan in aloe gel that could increase the number of macrophages (Sierra-GarcÃa et al., 2014). The results of this study showed that there was reduction in number of macrophages in the treatment group than negative control group on the 14th and 21st day after injury. Souza et al. (2017) reported decrease in the number of macrophages in burn rats treated with 1% silver sulfadiazine.\n\nIn this study, both the 3rd and 14th days after injury demonstrated higher number of fibroblasts in the treatment group than negative control group. This might have happened because of interaction between glucomannan in aloe gel and gibberellin, a growth hormone, with growth factor receptors on fibroblast. This interaction stimulated fibroblast activity and proliferation that could increase collagen synthesis (Chithra et al., 1998; Surjushe et al., 2008). Besides that, there was also acemannan in aloe gel which could increase the proliferation of fibroblasts (Xing et al., 2014). Fibroblasts are cells that will increase its migration during proliferation phase of wound healing. Therefore, this study showed highest number of fibroblasts on the 14th day after injury in the treatment group which was earlier than the negative control group that had the highest number on the 21st day after injury. At some point during the proliferation phase, there may be apoptosis of fibroblast when collagen matrix has filled the wound cavity (Gurtner & Wong, 2013). Therefore, there was reduction in the number of fibroblast on the 21st day after injury in the treatment group.\n\nRe-epithelialization occurs right after injury that consists of migration, proliferation and differentiation of keratinocytes (Gurtner & Wong, 2013; Isrofah and Afandi, 2015). Acemannan could stimulate fibroblast to release keratinocyte growth factor-1 (KGF-1) that could hasten the re-epithelialization process by keratinocyte which could increase the epidermal thickness (Jettanacheawchankit et al., 2009). The result of this study showed that there was an increase in the epidermal thickness in both, negative control and treatment groups from the 3rd day to 21st day after injury. But the treatment group had thicker epidermis than negative control group that might be caused by acemannan. An in vitro study conducted by Teplicki et al. (2018) demonstrated that Aloe vera could speeded up the migration and proliferation of keratinocyte. Another study conducted by Atiba et al. (2015) showed that Aloe vera could enhance the re-epithelialization process in corneal alkali burn in normal and diabetic rats.\n\n\nConclusion\n\nThis study showed that Aloe vera extract gel could accelerate the healing process of burns in rats.\n\n\nData Availability\n\nFigshare: Effect of Aloe Vera in post-burn skin repair in rats\n\nhttps://doi.org/10.6084/m9.figshare.17194973.v1\n\nFigshare: ARRIVE checklist for âEffect of Aloe Vera in Post-Burn Skin Repair in Ratsâ (Aulia and Pane, 2022)\n\nhttps://doi.org/10.6084/m9.figshare.17194973.v1\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nAuthor Contributions\n\nAulia L: Conceptualization, Formal Analysis, Investigation, Writing â Original Draft Preparation; Pane YS: Conceptualization, Formal Analysis, Writing â Original Draft Preparation, Writing â Review & Editing",
"appendix": "Acknowledgement\n\nThe authors give their appreciation and thanks to the Pharmacology Laboratory of the Faculty of Medicine, Universitas Sumatera Utara for allowing them to use the facility for collecting data for this research.\n\n\nReferences\n\nABA: Advanced Burn Life Support Course Provider Manual. American Burn Asociation; 2018.\n\nABA: 2019 ABA NBR Annual Report - American Burn Association. 2019. accessed 9 April 2021. Reference Source\n\nAtiba A, Wasfy T, Abdo W, et al.: Aloe vera gel facilitates re-epithelialization of corneal alkali burn in normal and diabetic rats. Clin. Ophthalmol. 2015. Publisher Full Text\n\nAulia L, Pane YS: Effect of Aloe vera extract in post-burn skin repair in rats. Figshare Dataset. 2022.\n\nChaby G, Viseux V, Poulain JF, et al.: Topical silver sulfadiazine-induced acute renal failure. Annales de Dermatologie et de Venereologie. 2005; 132: 891â893. PubMed Abstract\n\nChithra P, Sajithlal GB, Chandrakasan G: Influence of Aloe vera on collagen characteristics in healing dermal wounds in rats. Mol. Cell. Biochem. 1998; 181: 71â76. Publisher Full Text\n\nFederer WY: Experimental Design, Theory and Application. New York: Mac.Millan; 1963; 544. Reference Source\n\nFuller FW, Engler PE: Leukopenia in non-septic burn patients receiving topical 1% silver sulfadiazine cream therapy: A survey. J. Burn Care Rehabil. 1988; 9: 606â609. PubMed Abstract | Publisher Full Text\n\nGazali A, Ibnu I, Suriah: Perilaku Pencarian Pengobatan Terhadap Kejadian Penyakit Malaria Pada Suku Mandar Di Desa Lara Kecamatan Karossa Kabupaten Mamuju Provinsi Sulawesi Barat. Fakultas Kesehatan Masyarakat Universitas Hasanudin. 2013; 1â13.\n\nGurtner GC, Wong VW: Wound Healing: Normal and Abnormal. Grabb and Smithâ s Plastic Surgery. Lippincott Williams & Wilkins; 7th edn2013.\n\nIsrofah S, Afandi M: Efektivitas Salep Ekstrak Daun Binahong (Anredera Cordifolia (Ten) Steenis) Terhadap Proses Penyembuhan Luka Bakar Derajat 2 Termal pada Tikus Putih. Muhammadiyah Journal of Nursing. 2015.\n\nJettanacheawchankit S, Sasithanasate S, Sangvanich P, et al.: Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing. J. Pharmacol. Sci. 2009; 109: 525â531. PubMed Abstract | Publisher Full Text\n\nKristyaningsih P: Efektivitas Lidah Buaya (Aloe Vera) Dan Daunsirih (Piper Betle Linn) Terhadap Kesembuhan Luka Bakar Derajat Ii Pada Tikus Putih (Rattus Norvegicus Strain Wistar) Di Peternakan Tikus Sidomulyo Kediri. Jurnal Ilmu Kesehatan. 2016; 5: 114â121.\n\nMaulana RA: Faktor Resiko yang Berperan pada Mortalitas Penderita Luka Bakar Rawat Inap di RSUPH Adam Malik Medan dari Tahun 2011-2014. Universitas Sumatera Utara; 2014.\n\nMenkes.: Pedoman Nasional Pelayanan Kedokteran - Tata Laksana Luka Bakar. Keputusan Menteri Kesehatan Republik Indonesia. 2019.\n\nReinke JM, Sorg H: Wound repair and regeneration. Eur. Surg. Res. 2012; 49: 35â43. Publisher Full Text\n\nRussell WMS, Burch RL: The Principles of Humane Experimental Technique. London: Methuen& Co.Ltd; 1992. (Reissued: Universities Federation for Animal Welfare, Herts, England.) 1959. Reference Source\n\nSierra-GarcÃa GD, Castro-RÃos R, González-Horta A, et al.: Acemannan, an extracted polysaccharide from Aloe vera: A literature review. Nat. Prod. Commun. 2014; 9: 1934578X1400900. Publisher Full Text\n\nSmolle C, Cambiaso-Daniel J, Forbes AA, et al.: Recent trends in burn epidemiology worldwide: A systematic review. Burns. 2017; 43: 249â257. PubMed Abstract | Publisher Full Text\n\nSouza HR, De Azevedo LR, Possebon L, et al.: Heterogeneity of mast cells and expression of Annexin A1 protein in a second degree burn model with silver sulfadiazine treatment. PLoS ONE. 2017; 12: e0173417. PubMed Abstract | Publisher Full Text\n\nSurjushe A, Vasani R, Saple D: Aloe vera: A short review Indian J. Dermatol. 2008; 53: 163â166. PubMed Abstract | Publisher Full Text\n\nTeplicki E, Ma Q, Castillo DE, et al.: The effects of aloe vera on wound healing in cell proliferation, migration, and viability. Wounds. 2018; 30: 263â268. PubMed Abstract\n\nWHO: WHO|Burns. 2018. accessed 29 May 2021. Reference Source\n\nXing W, Guo W, Zou CH, et al.: Acemannan accelerates cell proliferation and skin wound healing through AKT/mTOR signaling pathway. J. Dermatol. Sci. 2014; 79: 101â109. Publisher Full Text"
}
|
[
{
"id": "123296",
"date": "28 Feb 2022",
"name": "Gusbakti Rusip",
"expertise": [
"Reviewer Expertise Physiology human",
"exercise",
"and much research-related use plants as herbal medicine"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Lusiana Aulia and Yunita Sari Pane presents the intervening Effect of Aloe vera extract in post-burn skin repair in rats. In general, I find this manuscript to make an important contribution in developing palatable and superior bioactive compounds in Aloe vera containing various active ingredients that help the wound healing process, such as glucomannan and acemannan. However, I have outlined below some of the issues that need to be addressed prior to indexing, and some minor comments and suggestions to further improve the quality of the manuscript.\nMajor\nIn the abstract and introduction, the author states that one of the objectives of this study is to improve post-burn skin with the intervention of Aloe vera extract. However, histopathological description of the healing process of burns is not presented and discussed.\n\nThe method part is not clear. It is not clear the analytical method for macrophage and fibroblast counts and the analytical formula is not specified, the authors need to explain it. Please clarify and make changes to the methods section as appropriate.\n\nIn the discussion section, it is not explained how this study shows a synergistic interaction between changes that occur on days 3, 14 and 21 and between a number of macrophages and fibroblasts in figures 1, 2 and 3 with no visible histopathologic changes. It's better to call it the calculation score.\n\nMinor\nIn the method section, how much Aloe vera contains? Explain the total solids content of the gel material used in the study.\n\nIn the results section, a description of the histopathological picture is explained in relation to the analysis results from figures 1,2 and 3. It would be easier to explain in the discussion section.\n\nIn the discussion section, please explain clearly the bioactive compounds contained in Aloe vera other than glucomannan and acemannan. Are there other active ingredients? What do the authors mean treatment days 14 and 21? Describe the histopathological picture with figures 1,2 and 3.\n\nIn the introduction and abstract, Aloe vera intervention changes macrophages, fibroblasts and increases bioactive compounds and their potential for healing functional burns. The conclusion states that it is a potential pharmaceutical product. Please be consistent.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "7932",
"date": "10 Mar 2022",
"name": "Yunita Sari Pane",
"role": "Author Response",
"response": "Dear Prof Gusbakti Rusip, I really appreciate your willingness to review my manuscript. Here I explain more information to improve understanding in this article. Major: We have assessed entirely during the present study, but I couldn't document all due to lack of memory byte card on the camera, So I took a few photos that can represented it and can be seen in figshare (https://doi.org/10.6084/m9.figshare.17194973.v1)  We used the ANOVA test because in this study we used more than two groups with parametric data types  We have presented and correlated the changes that occur on days 3,14,21 in macrophages and fibroblasts that could be seen in Figures 1 and 2 and had been discussed in the discussion section. Minor: Aloe vera contained 10% of the total weight of the gel (see gel preparation)  In journal writing, repetition of words should be avoided, therefore we chose to explain in detail the number and comparison between groups in the results section. While in the discussion section we explained the comparison of our findings with other studies  Aloe vera has active ingredients other than acemannan and glucomannan, but based on the literature that we read, it proved that they could play a role in burn repair.  We did not state that aloe vera can be used as a pharmaceutical product in the conclusion section (see part conclusion). We only stated that aloe vera could accelerate the healing process as proven by this study."
}
]
},
{
"id": "128998",
"date": "28 Apr 2022",
"name": "Fatima Ali",
"expertise": [
"Reviewer Expertise Stem cell"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor:\nThe authors mainly focuses on wound repair after burn injury but fail to show the result. Only the data of fibroblast and macrophages did not justify the whole questions? To answer this question author must show picture of wound healing, histopathological picture, wound healing area,\n\nHow authors say that the size of 2Ã2 cm2 is reduced? Author must show the data about the wound healing in term of wound healing picture and wound healing area?\n\nImages of a H&E staining should be provided and correlate it with number of fibroblast and other mentioned cells.\n\nImprove the scientific language.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "8450",
"date": "29 Jul 2022",
"name": "Yunita Sari Pane",
"role": "Author Response",
"response": "Dear Fatima Ali, Thank you for the comments on our manuscript. I would like to highlight some particulars regarding the work we have done according to the comments: We have revised the results section regarding histopathological images as microscopic parameters. So, we don't need to show a picture of wound healing (macroscopic parameters), because it isnât in accordance with the aim of this study i.e: mean number of macrophages, number of fibroblasts, and epidermal thickness.  In this study, there is no statement of a reduction in burns. As for the information about 2x2cm2, it is the area of ââââthe wound that was determined in this study ( see in wound inflection part of the method).  In the revision, we have added histopathological images of macrophages, fibroblasts, and epidermal thickening. the explanation histopathological picture can be correlated with pictures 1, 2, and 3 (see results section).  Thank you for your suggestion, we will try to improve our ability in scientific language."
}
]
}
] | 1
|
https://f1000research.com/articles/11-168
|
https://f1000research.com/articles/11-1145/v1
|
07 Oct 22
|
{
"type": "Research Article",
"title": "Qualitative analysis of HIV and AIDS disease transmission: impact of awareness, testing and effective follow up",
"authors": [
"Oluwakemi E. Abiodun",
"Olukayode Adebimpe",
"James Ndako",
"Olajumoke Oludoun",
"Benedicta Aladeitan",
"Michael Adeniyi",
"Olukayode Adebimpe",
"James Ndako",
"Olajumoke Oludoun",
"Benedicta Aladeitan",
"Michael Adeniyi"
],
"abstract": "Background: Since the early 1980s, human immunodeficiency virus (HIV) and its accompanying acquired immunodeficiency syndrome (AIDS) have spread worldwide, becoming one of the world's major global health issues. From the beginning of the epidemic until 2020, about 79.3 million people became infected, with 36.3 million deaths due to AIDS illnesses. This huge figure is a result of those unaware of their status due to stigmatization and invariably spreading the virus unknowingly. Methods: Qualitative analysis through a mathematical model that will address HIV unaware individuals and the effect of an increasing defaulter on the dynamics of HIV/AIDS was investigated. The impact of treatment and the effect of inefficient follow-up on the transmission of HIV/AIDS were examined. The threshold for the effective reduction of the unaware status of HIV through testing, in response to awareness, and the significance of effective non-defaulting in treatment commonly called defaulters loss to follow-up as these individuals contribute immensely to the spread of the virus due to their increase in CD4+ count was determined in this study. Stability analysis of equilibrium points is performed using the basic reproduction number $R_0$, an epidemiological threshold that determines disease eradication or persistence in viral populations. We tested the most sensitive parameters in the basic reproduction numbers. The model of consideration in this study is based on the assumption that information (awareness) and non-stigmatization can stimulate change in the behaviours of infected individuals, and can lead to an increase in testing and adherence to treatment. This will in turn reduce the basic reproduction number, and consequently, the spread of the virus. Results: The results portray that the early identification and treatment are inadequate for the illness to be eradicated. Conclusions: Other control techniques, such as treatment adherence and effective condom usage, should be investigated in order to lessen the disease's burden.",
"keywords": [
"HIV/AIDS",
"infection-free equilibrium",
"defaulter lost to follow-up",
"endemic equilibrium",
"next generation matrix",
"basic reproduction number",
"stability."
],
"content": "1. Introduction\n\nHuman immunodeficiency virus (HIV) is a sexually transmitted infection (STI) and a blood-borne illness in humans with a wide range of clinical manifestations.1,2 HIV and its accompanying acquired immune deficiency syndrome (AIDS) have spread rapidly around the world since its discovery in the early 1980s, and it remains the worldâs most serious global health and development challenge. There is, however, a global devotion to avoiding new infections and making sure that all patients diagnosed have access to treatment. In addition, 79.3 million individuals have been infected with HIV since the pandemic began, with 36.3 million people dying due to AIDS diseases. About five million individuals contracted HIV for the first time in 2003, the largest number in any one year since the pandemic began.3 Globally, the figure of persons living with HIV/AIDS has risen from 35 million in 2001 to 37.7 million in 2020, with around 3 million people dying from the illness in that year.4,5 Around 84 percent [68 â 98 percent] of HIV-positive persons in the globe know their status in 2020, the remaining 16 percent (about 6 million people) [4.8 million-7.1 million] need to be tested for HIV. HIV testing is an important initial step in HIV prevention, treatment, care, and support.6,7 Under Sustainable Development Goal 3, the international community pledged to work to end the AIDS pandemic by 2030. While progress has been made, it has been inconsistent, and the intermediate targets of â90-90-90â have been missed.7,8 New diseases continue to wreak havoc on communities and undermine vital socioeconomic infrastructure all across the planet. According to the United Nations Joint Program on HIV and AIDS, the number of HIV-positive people in 2021 was 37.6 million, up from 33.2 million in 2010.9 1.5 million [1.1 million-2.1 million] people contracted HIV for the first time in 2020, 690,000 [480,000-1 million] people died of AIDS-related illnesses, and antiretroviral medication was available to 27.4 million [26.5 million-27.7 million] patients in December 2020, up from 7.8 million [6.9 million-7.9 million] in 2010.9â11 HIV can be spread horizontally or vertically from one infected individual to another. Horizontal HIV transmission occurs when an individual comes into direct contact with an HIV-positive person, including sexual contact, or when they use a needle and syringe that has recently been utilized by a HIV-positive individual. Contrastingly, vertical transmission occurs when the virus is passed directly from an infected mother to her pregnant or newborn child.12 HIV/AIDS transmission dynamics has piqued the interest of applied mathematicians, epidemiologists13â16 and biologists17â22 due to the diseaseâs worldwide menace. Various improvements have been made to May and Andersonâs early models,23â25 and particular issues have been discussed by researchers.12,26â48 In Lu et al. 202027 fostered a compartmental model for the yearly revealed HIV/AIDS MSM in the Zhejiang Region of China between 2007 to 2019 and anticipated that 90 percent of people tested for HIV/AIDS will have received treatment by 2020, while the screened extent will remain as low as 40 percent, and that antiretroviral treatment (ART) can actually control the transmission of HIV, even within the sight of medication opposition. In Rana and Sharma, 202030 presented a simple Likely to be exposed-Infected (i.e.SI) form of HIV/AIDS mathematical model, in view of the supposition that changing from an AIDS-infected to an HIV-infected individual is conceivable, in order to understand disease dynamics and develop strategies to reduce or control disease transmission among individual. Mushanyu32 built a mathematical model for HIV acquisition using nonlinear ordinary differential equations to analyse the influence of delayed HIV diagnosis on the transmission of HIV in the year 2020. To prevent HIV from spreading further, the researchers advocated for early HIV treatment and the expansion of HIV self-testing initiatives, which would allow more people who have not been tested for HIV to learn their status. Teng12 proposed and investigated a time-delay compartmental framework for HIV transmission in a sexually active cohort with press coverage, a disease that can result to a developed phase of infection known as acquired immunodeficiency syndrome (AIDS), as well as vertical transmission in the enrollment of people infected in 2019.33 Saad et al. (2019) developed and considered an HIV+ mathematical model with the next generation matrix, the infection-free and endemic equilibrium points were identified, and the basic reproduction ratio R0 was determined. The Lyapunov function was utilized to analyze the equilibriaâs global stability, and it was observed that the equilibriaâs stability is reliant on the magnitude of the fundamental reproduction ratio.37 developed an HIV/AIDS epidemic model with a generic nonlinear rate of occurrence and therapy, was able to obtain the basic reproductive number R0 using the next generating matrix technique.\n\nResearchers have employed numerous tools to manage and eradicate HIV/AIDS diseases.3,11,12 These studies revealed that awareness creation/information can help to control the disease burden but cannot eliminate the disease. Furthermore, there are other techniques and tools available that can be applied to study the dynamics of disease transmission and to provide suitable control interventions. The use of mathematical modeling is foremost among these techniques.16â19 Although many articles20 have studied the impact of different controls; however, none of them have incorporated human behavior in response to information. Hence, this study identifies the threshold for effective reduction of HIV/AIDS, as a result of HIV unaware individuals and consequent effective follow up in the use treatment.\n\nThe following is the structure of the paper: Section 2 describes the model, while Section 3 examines the modelâs basic features, the basic reproduction number, and equilibrium points. Section 4 employs parameter sensitivity index on the reproduction number to conduct a stability study of the equilibria (local and global), and the findings are generated from numerical simulations of data from previously published studies in Section 5. Finally, the research is examined and completed in Section 6.\n\n\n2. Model formulation and description\n\nA mathematical model on the mechanisms of horizontal and vertical transmission of HIV/AIDS was developed, by incorporating the effect of testing, defaulter lost to follow-up on treatment, and effective use of condom on the existing model. The model is available from GitHub and is archived with Zenodo.66 The model is depicted schematically in Figure 1. The model contains six (6) state variables, namely: Susceptible, (S), representing people who are likely to become infected with HIV; Unaware HIV infectives, (HU), Aware HIV infectives (HA), Treated HIV infectives, (HT); AIDS individuals (AA) and AIDS on treatment individuals (AT). The rate of effective contact with HIV-positive people either by immigration or emigration is given by Î. A percentage of newborns get infected with HIV during birth at a rate of (1 â ζ) and are therefore directly enrolled into the unaware infected population HU, at a rate ζÎ, with 0 †ζ †1. λH=c1âÏΟβ1HU+β2HA+β3AAN is the HIV transmission contact rate. Parameter c represents the average number of sexual partners acquired by people who is vulnerable to HIV annually. In order to simulate the influence of condom usage as a significant preventive intervention, the amount of condom protection (usage and effectiveness) is given as ÏΟ[0, 1] based on assumption. If Ο = 0, condom use provides no protection, but Ο = 1 denotes complete protection, where Ï is the condom use. The parameters β1, β2 and β3 account for the HIV transfer rates between persons at risk and (HIV unaware, HIV aware and full blown AIDS) infectives individuals, respectively. Both the HIV-infected and the AIDS-infected groups are thought to be active in the spread of HIV/AIDS amongst susceptible. Because infected patients with AIDS symptoms have a greater viral load than HIV positive people (pre-AIDS) in the HU and HA classes, and because viral load and infectiousness have a positive connection, we must have β1 < β2 < β3. There is an evidence to suggest that individuals who know their HIV status HA change their sexual behavior (i.e. adopt safer-sex practices), resulting in reduced transmission.25 Most HIV pandemic models disregard the role of AIDS patients in HIV transmission by applying simplistic assumptions such as AIDS death being immediate or AIDS patients being incapable of mingling and gaining new sex partners. However, epidemiological data shows that AIDS patients participate in hazardous sexual activities, such as seldom wearing condoms or having several sex partners.61 As shown in the findings of21 a research of HIV-1-infected transfusion men and their women sex partners, severe AIDS patients are more likely to infect their partners than non-advanced immuno-compromised receivers.62 also reported similar findings. HIV-positive individuals with and without AIDS signs are likely to have access to antiretroviral therapy (ART). Unaware HIV-infected persons, HU, progress to the category of aware HIV infection HA, after testing at a rate of α, while unaware infected individual who did not go for testing progress to stage IV of AIDS, AA; at a rate of Ï. HIV-infected aware people with no symptoms of AIDS; HA, proceed to the group of HIV infection under ART therapy, HT, whereas HIV-infected people with AIDS symptoms, AA, are treated for AIDS at a rate of Ξ2 on reaching the class of AT. We presume that HIV-infected people on treatment do not spread the virus.49,50 HIV-infected people who are receiving therapy but do not have AIDS symptoms, HT, who default during treatment and become resistant to drug, will return to the HIV-infected aware individuals, HA, and that HIV-infected persons with AIDS symptoms, AA, who default during treatment in class AT, become re-infected with HIV with symptoms of AIDS individuals, AA, at a rate Ï
1 and Ï
2 respectively.51 It is assumed that only HIV-infected people with AIDS symptoms, AA and AT, die of AIDS-related causes at a rate of da. The following mathematical model is based on these assumptions and that the system has a natural death in each class at a rate ÎŒ.\n\nIn order to contribute to the arduous aim of ending it by 2030 there is need to foresee the epidemicâs behaviour. One of the most significant tools weâll utilize to attain our aim is mathematical modeling of HIV infection. Based on,52 the following model was developed by the inclusion of AIDS on treatment compartment (by considering treatment of both individual not showing and showing symptoms of AIDS), individual who fall-out of treatment, considering AIDS individual are able to transmit infection, condom use to control transmission rate and average number of sexual partners acquired on force of infection. A system of ordinary differential equations (ODEs) can be used to express the mathematical equations that correspond to the schematic diagram:\n\n\n3. Model investigation\n\nAll of the parameters in the model are considered to be non-negative. System (1), on the other hand, keeps track of the human populace, hence, the state variables are always positive for all time t ⥠0. Thus, the total human populace is given as\n\nHere equation (1) is changing at a rate\n\nIn the non-existence of infection i.e for HU = HA = HT = AA = AT = 0 we have,\n\nWe must have (6) by separating the variables of differential inequality.\n\nIntegrating the above equation we have\n\nFrom (7) we have\n\nAs tââ,0â€Ntâ€ÎÎŒ\n\nAs a result, the system (1) feasible solutions set enters the region.\n\nThis section discusses the positivity of the solutions, which describes the systemâs non-negativity of solutions (1).\n\nLemma 1: S(t) ⥠0, HU(t) ⥠0, HA(t) ⥠0, HT(t) ⥠0, AA(t) ⥠0, AT(t) ⥠0 and N(t) ⥠0 satisfied by the solutions of system (1) with initial conditions (2) for all t ⥠0. The region Ωâââ+06 is positively invariant and attracts in terms of system (1).\n\nProof: Take a look at the first equation in (1)\n\nprovided ÎζHU+λH+ÎŒ<â\n\nAs a result, S ⥠0\n\nLikewise, for system (1)âs second equation, we have\n\nprovided α+Ï+ÎŒ<â\n\nHence, HU ⥠0\n\nsimilarly it can be shown that HA ⥠0, HT ⥠0, AA ⥠0, AT ⥠0 for all t > 0\n\nThus the solutions S, HU, HA, HT, AA, AT remain positive forever.\n\nThe model (1) has exactly one disease-free equilibrium (DFE) point and the equilibrium point E0 is given by S0HU0HA0HT0AA0AT0=ÎÎŒ00000. In the absence of infection, the total population changes in proportion to the ratio of recruitment rate to the death rate.\n\nThe total population dynamics can be altered when an individual with an HIV/AIDS is introduced into a population. For the endemic equilibrium, there is an existence of infection hence HUâ HAâ HTâ AAâ ATâ 0. It is denoted by E*. Setting equation (1a-1f) equal to zero which exist when R0 > 1 we have\n\nM1 = α + Ï + ÎŒ, M2 = Ξ1 + ÎŒ, M3 = Ï
1 + ÎŒ, M4 = Ξ2 + da + ÎŒ, M5 = Ï
2 + da + ÎŒ.\n\nTheorem 1: There exists a positive endemic equilibrium if R0 > 1\n\nReference 53 presented a better method for determining R0 which was an improved technique of solving the reproduction number firstly developed by Ref. 54 that is widely accepted because it represents the biological meaning of R0. By considering only the infective classes, we were able to obtain the systemâs (1) basic reproduction number, R0, which is the spectral radius (Ï) of the next generation matrix, NGM, i.e.R0=ÏFVâ1. The rate of emergence of new infections in compartments i, while V denotes the rate of transfer of individual into and out of the compartment i by all other means. Where F and V are the m à m matrices defined as:\n\nF=âFixoâxjandV=âVixoâxjwithiâ€i,jâ€m\n\nF is non-negative and V is non-singular matrix.\n\nThen,\n\nThe model reproduction number, denoted by R0 is thus given byR0=ÏFVâ1=R=R1+R2+R3 , the spectral radius of the NGM FVâ1.\n\nHere,\n\n\n4. Equilibria stability analysis\n\nTheorem 2: For all R0, the disease-free equilibrium E0 exists, and it is locally asymptotically stable for R0 < 1 and unstable otherwise.\n\nProof: The resulting matrix from linearized model dxdt=AX, where X=x1x2x3x4x5x6T,x1x2x3x4x5x6âR+6 , and\n\nThe resulting Jacobian matrix of (14) at E0 is\n\nfrom (15) the first three eigenvalues are given as λ1=âÎŒ,λ2=âv1+ÎŒ,λ3=âΞ1+ÎŒ and the roots of the resulting quadratic equation is obtained as:\n\nBecause all parameters of the model are assumed to be positive, λ4 < 0, λ5 < 0, λ6 < 0. Evidently, if R0 < 1, the roots of f(λ) have negative real parts, implying that E0 is locally asymptotically stable (LAS) when R0 < 1; if R0 > 1, the roots of f(λ) are real and some are positive, implying that E0 is unstable.\n\nTheorem 3: If R0 < 1, the disease free equilibrium is asymptotically stable globally for system (1).\n\nProof: The comparison theorem, as demonstrated by Ref. 55 proves the global stability of the disease-free equilibrium. We rename the infected class: dxdt=FâVXâJX,X=HUHAHTAAAT where,\n\nThen all of the matrix F â V eigenvalues have negative real parts, i.e So that\n\nEquation (20) has four (4) negative roots by Descartes rule of signs if\n\nSince Stâ€ÎÎŒ in the invariant set, J is a non-negative matrix. Hence, it follows that\n\nWhen R0 < 1, the eigenvalues of the matrix F â V are negative. As a result, the linearized differential equation is stable whenever R0 < 1 is positive. Since HUHAHTAAATâ00000 as tââ. According to the comparison theorem,HUHAHTAAATâ00000 as tââ. Substituting HU = HA = HT = AA = AT = 0 in (1) gives StâS0 as tââ. Thus, SHUHAHTAAATâS000000 as tââ for R0 < 1. Thus, E0 is globally asymptotically stable if R0 < 1.\n\nTheorem 4: The endemic steady state EâSâHuâHAâHTâAAâATâ of the model is locally asymptotically stable (LAS) If R0 > 1.\n\nProof: We must now demonstrate the local stability of the endemic steady state. Assume R0 > 1.\n\nThe Jacobian matrix for the variables of system (1) is computed in the proof of Theorem 2 as in (14).\n\nHence, for the endemic equilibrium SâHUâHAâHTâAAâATâ) , the Jacobian matrix and the determinantal equation at the endemic equilibrium is given as matrix in (15)\n\nClearly, the equation reduces to:\n\nThe first four eigenvalues of (21) are given as:\n\nThe eigenvalue of the remaining 2 à 2 is obtained from the characteristics equation below:\n\nThe determinants of the characteristic polynomial from (22) yield the following result:\n\nPolynomials of order 2 satisfy the Routh-Hurwitz criterion, We know that f(λ) = 0 using Routh-Hurwitz criterion polynomials of order 2 is stable if and only if both coefficients in (22) satisfy the following conditions: ai > 0 From Eq. (22) the condition is satisfied. Therefore, EE is locally asymptotically stable.\n\nTheorem 5: when R0<1, the equations of the model have a positive distinct endemic equilibrium, which is said to be globally asymptotically stable.\n\nProof: Considering the Lyapunov function, which is defined as\n\nAt equilibrium\n\nThus\n\nHence, by Lasalle theorem, the equilibrium is globally asymptotically stable in the feasible region R+6.\n\nKnowing the relative relevance of the different factors involved in HIV transmission and prevalence is vital for deciding how effectively to minimize human morbidity and mortality rate due to HIV infections. Sensitivity analysis is performed in this sub-section to assess the resilience of factors that have a strong impact on the basic reproduction number, R0, so that suitable intervention strategies may be implemented.\n\nThe effect of HIV testing and treatment on HIV/AIDS dynamics was studied using the elasticity of ReH with respect to α and Ξ. Using the method described in57,64,65 to compute the elasticity58 of ReH with respect to α and Ξ as shown in Equation (25)\n\nInterpretation of the sensitivity indices\n\nTable 1âs sensitivity indices are read as follows: Positive indices indicate that the corresponding basic reproduction number increases (decreases) as those parameters increase (decrease). Negative indices, on the other hand, indicate that increasing (decreasing) those parameters reduces the associated basic reproduction number (increases).\n\nThe endemicity of HIV infection increases when the values of βi, i = 1, 2, 3, Ï
, and c are increased; when the values of alpha and mu are decreased, the endemicity of HIV infection decreases.\n\nAs a result, interventions should aim to reduce the annual average number of sexual partners acquired, c, the number of defaulters lost to follow-up, Ï
, and the likelihood of HIV transmission per sexual contact, βi, i = 1, 2, 3, because the rate of progression from HIV to AIDS is increasing, Ï, indicates rapid progression to AIDS. In addition, effective condom use should be mandated as a precautionary measure to reduce the rate of HIV/AIDS transmission.\n\n\n5. Numerical simulation\n\nTo affirm the modelâs theoretical prognosis, simulation studies of the system (1) are run with the estimated parameter values listed below:\n\nSimulation 1. Take into account the parametric data in Table 2 c = 3, Ï = 0, Ο = 0, β1 = 0.050, β2 = 0.055, β3 = 0.060, ÎŒ = 0.2, Î = 29, α = 0.7, Ï = 0.322, ζ = 0.02, Ï
1 = 0.0169, Ï
2 = 0.0169, Ξ1 = 1.6949, Ξ2 = 1.6949, da = 0.0333: Hence, R0 = 0.698 and the infection-free equilibrium is (145.000;0;0;0;0;0): We can see in Figure 2 that by changing the initial values, the solution trajectories intersect to (145.00;0;0;0;0;0): This confirms the fact that if R0 < 1, the virus-free equilibrium is globally asymptotically stable:\n\nSimulation 2. Let c = 6, Ï = 0, Ο = 0, β1 = 0.080, β2 = 0.085, β3 = 0.090, ÎŒ = 0.2, Î = 29, α = 0.7, Ï = 0.322, ζ = 0.02, Ï
1 = 0.0169, Ï
2 = 0.0169, Ξ1 = 1.6949, Ξ2 = 1.6949, da = 0.0333: Hence, R0 = 2.197. Moreover, the endemic equilibrium is (64.197;13.225;5.251;41.035;2.348;15.905): We can see in Figure 3 that by changing the initial conditions, the solution trajectories intersect to (64.197;13.225;5.251;41.035; 2.348;15.905): This proves Theorem 5: if R0 > 1, the endemic stability is globally stable.\n\nSimulation 3 depicts the distribution of individual proportions over time in various classes where there are no new infected children ζ or recruitment Î, and contact c i.e. taking c = 0, ζ = 0, Î = 0 when Ï = 1 and Ο = 1, (condom usage and effectiveness)i.e when there is full protection keeping every other values at endemic equilibrum constant, the value of R0 = 0.\n\nThe impact of perinatal transmission in the system, i.e. the incidence of new recruits of infected children directly into the infective group, is pointedly demonstrated in simulation 4.\n\nFigure 5(a) shows that as the proportion of infected newborns (ζ) rises, so does the proportion of the general population who is unaware. Figure 5(b) shows that increasing the value of (ζ) causes the proportion of the AIDS population to decrease over time, then raise until it reaches its stable state. As a result, if newborns infected with the virus are treated, the total infective group will be better controlled, minimizing the AIDS individuals. Figure 5(c) shows that as the number of infected children born rises, so does the treated populace.\n\nA. Variation of Unaware HIV population for different values of ζ. B. Variation of Aware HIV population for different values of ζ. C. Variation of HIV on Treatment population for different values of ζ. D. Variation of AIDS population for different values of ζ. E. Variation of AIDS on Treatment population for differnet values of ζ.\n\nThe effect of defaulters on treatment lost to follow-up in the model is examined in simulation 5.\n\nFigure 6(a) shows that as the rate of defaulters (Ï
) increases, so does the proportion of the population that is aware, whereas the proportion of HIV patients on treatment decreases (b). Figure 6(c) shows how increasing upsilon causes the proportion of the AIDS population to increase over time while decreasing the proportion of the AIDS population on treatment until equilibrium is reached. As a result, if the HIV-aware infected population follows adheres therapy, the infectious individual as a whole would then remain under control, lowering the HIV-aware and AIDS number of individuals.\n\nA. Variation of HIV Aware population for different values of Ï
. B. Variation of HIV on Treatment population for different values of Ï
. C. Variation of AIDS population for different values of Ï
. D. Variation of AIDS on Treatment population for different values of Ï
.\n\nThe increasing effect of testing and treatment on the model is examined in simulation 6.\n\nFrom Figure 7(a-d), it is observed that if testing rate and treatment rate is increase,the unaware HIV decrease,while aware HIV and AIDS individual decrease with time due to treatment. Furthermore, the susceptible individual increases, and as treatment increases, so does the population of HIV and AIDS patients on treatment. As a result, increasing HIV screening and treatment is the first procedure to UNAIDSâ 90-90-90 aspirations.\n\nA. Proporation of Population when α = 0.7 and Ξ = 1.6949. B. Proporation of Population when α = 0.9 and Ξ = 2.6949. C. Proporation of Population when α = 1.5 and Ξ = 4.6949. D. Proporation of Population when α = 1.9 and Ξ = 9.6949.\n\nFigure 8 shows the effect of treatment fall out on the reproduction number. When the number of infected individual on treatment that fallout is 19.8 percent then R0 = 0.041. The linear graphical representation also revealed that if 40.1 percent of the population drops out of treatment, the reproduction number rises to 0.043. This simply means that, as defaulters lost to follow-up increase, the reproduction number also increases. Hence, reducing high-risk habits, mainly through education, is the most effective way to reduce the overall number of HIV/AIDS patients.\n\n\n6. Conclusions and recommendations\n\nThis study investigated the effect of testing and ART on the vertical and horizontal transmission dynamics of HIV/AIDS infection using an improved compartmental model and the dynamics theory of SI infectious diseases.\n\nReducing high-risk behaviours, primarily through education on the importance of HIV/AIDS status awareness and treatment adherence, is the best option for reducing the total number of HIV/AIDS patients.\n\nIncreased HIV testing is the first step toward UNAIDSâs 90-90-90 objectives, although many countries still face significant obstacles in attaining this goal. Early detection allows for prompt antiretroviral therapy, which lowers HIV viral load and hence slows the transmission of the virus. We believe that increasing HIV/AIDS diagnosis rates will increase the number of HIV/AIDS patients treated in the short term but decrease the number in the long term. WHO advises HIV self-testing as a complementary strategy,59 which can improve the efficiency of HIV testing.60\n\nThe current research showed that these intervention strategies are effective in combating the HIV/AIDS epidemic. This also emphasizes the need of behavioral and biologic therapies in preventing HIV transmission among pregnant women. This study has flaws, as well. First, statistics on drug resistance may be skewed because not all treated patients are tested early on, and secondly, homosexual transmission was not included in the model. Finally, certain characteristics were chosen on the basis of assumptions and may not really reflect reality.\n\nIn conclusion, the model implies that, in addition to HIV testing, behavioural and biologic strategies, effective condom use, and stringent adherence to ART are required for HIV prevention among individuals and pregnant women. Even in the face of medication resistance, ART and effective condom use can successfully limit the transmission of HIV. The 90-90-90 strategy may not be sufficient on its own to end the global HIV/AIDS outbreak.\n\n\nData availability\n\nThe data in this article come from Mukandivire et al., 2010, Zu et al., 2016, Lu et al., 2020, and other assumed/estimated data.\n\n\nSoftware availability\n\nSource code available from: https://github.com/OE-Abiodun/release/tag/v3.1.2\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.6894864.66\n\nLicense: GNU General Public License v3.0",
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Equ. 2014; 2014(1): 1â17. Publisher Full Text\n\nLi Q, Cao S, Chen X, et al.: Stability analysis of an HIV/AIDS dynamics model with drug resistance. Discret. Dyn. Nat. Soc. 2012; 2012: 1â13. Publisher Full Text\n\nDaabo MI, Seidu B: Modelling the Effect of Irresponsible Infective Immigrants on the Transmission Dynamics of HIV/AIDS.2012; 3(1): 31â40.\n\nAl-sheikh S: Stability Analysis of an HIV/AIDS Epidemic Model with Screening.2011; 6(66): 3251â3273.\n\nNyabadza F, Mukandavire Z, Hove-Musekwa SD: Modelling the HIV/AIDS epidemic trends in South Africa: Insights from a simple mathematical model. Nonlinear Anal. Real World Appl. 2011; 12(4): 2091â2104. Publisher Full Text\n\nAkpa OM, Oyejola BA: Modeling the transmission dynamics of HIV/AIDS epidemics: An introduction and a review. J. Infect. Dev. Ctries. 2010; 4(10): 597â608. PubMed Abstract | Publisher Full Text\n\nCai L, Li X, Ghosh M, et al.: Stability analysis of an HIV/AIDS epidemic model with treatment. J. Comput. Appl. Math. 2009; 229(1): 313â323. Publisher Full Text\n\nMarks G, Crepaz N, Senterfitt JW, et al.: Meta-analysis of high-risk sexual behavior in persons aware and unaware they are infected with hiv in the united states: implications for hiv prevention programs. J. Acquir. Immune Defic. Syndr. 2005; 39: 446â453. PubMed Abstract | Publisher Full Text\n\nEshleman SH: HHS Public Access. Physiol. Behav. 2019; 176(3): 139â148. PubMed Abstract | Publisher Full Text\n\nPing L-H, Jabara CB, Rodrigo AG, et al.: HIV-1 Transmission during Early Antiretroviral Therapy: Evaluation of Two HIV-1 Transmission Events in the HPTN 052 Prevention Study. PLoS One. 2013; 8(9): e71557. PubMed Abstract | Publisher Full Text\n\nZhimin S, Dong C, Li P, et al.: A mathematical modeling study of the HIV epidemics at two rural townships in the Liangshan Prefecture of the Sichuan Province of China. J. Infect. Dis. Model. 2016; 1: 3â10.\n\nZu G, Mo I, Danbaba A, et al.: Mathematical Modelling for Screening and Migration in Horizontal and Vertical Transmission of HIV/AIDS.2016; 5(1): 4â10.\n\nvan den Driessche P , Watmough J: Reproduction numbers and subthreshold endemic equilibria for compartmental models of disease transmission. Math. Biosci. 2002; 180: 29â48. PubMed Abstract | Publisher Full Text\n\nDiekmann O, Hesterbeek JAP, Metz JAJ: On the definition and the computation of the basic reproduction ratio R 0 in models for infectious diseases in heterogeneous populations. J. Math. Biol. 1990; 28: 365â382. PubMed Abstract\n\nOkuonghae 2016.\n\nBrauer F, Castillo-Chaavez C: Mathematical models for communicable diseases, volume 84. SIAM.2012.\n\nChitnis N, Hyman JM, Cushing JM: Determining important parameters in the spread of malaria through the sensitivity analysis of a mathematical model. Bull. Math. Biol. 2008; 70(5): 1272â1296. PubMed Abstract | Publisher Full Text\n\nCaswell H: Construction, analysis, and interpretation. Sunderland:Sinauer;2001.\n\nWHO Guidelines on HIV Self-Testing and Partner Notification: Supplement to Consolidated Guidelines on HIV Testing Services. Geneva:World Health Organization;2016.\n\nTang W, et al.: What happens after HIV self-testing? Results from a longitudinal cohort of Chinese men who have sex with men. BMC Infect. Dis. 2019; 19: 807. PubMed Abstract | Publisher Full Text\n\nLansky A, Nakashima A, Jones J: Risk behaviors related to heterosexual transmission from HIV2 38 infected persons. Sex. Transm. Dis. 2000; 27: 483â489. PubMed Abstract | Publisher Full Text\n\nNicolosi A, Musicco M, et al.: Risk factors for woman-to-man sexual transmission 255 of the human immunodeficiency virus. J. Acquir. Immune Defic. Syndr. 1994; 7: 296â300. PubMed Abstract\n\nOBrien T, Busch M, et al.: Heterosexual transmission of human 257 immunodeficiency virus type 1 from transfusion recipients to their sex partners. J. Acquir. Immune Defic. Syndr. 1994; 7: 705â710.\n\nBrauer F, Castillo-Chaavez C: Mathematical models for communicable diseases. SIAM;2012; vol. 84.\n\nBrauer F, Castillo-Chavez C: Mathematical models in population biology and epidemiology. Springer;2001; vol. 40.\n\nAbiodun OE: OE-Abiodun/OE-Abiodun: F1000: HIV ONLY MODEL (v3.1.2). [Software] Zenodo.2022. Publisher Full Text"
}
|
[
{
"id": "152688",
"date": "18 Nov 2022",
"name": "Adedapo Loyinmi",
"expertise": [
"Reviewer Expertise Biological and computational Mathematics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article proposed a mathematical model for the transmission dynamics of HIV and AIDS considering three control/preventive strategies: the impact of awareness, testing and follow up. The work presented a six- compartmental model of HIV/AID which are Susceptible, (S), representing people who are likely to become infected with HIV; Unaware HIV infective, (HU), Aware HIV infective (HA), Treated HIV infective, (HT); AIDS individuals (AA) and AIDS on treatment individuals (AT). Analysis of the model showed that the model is positively invariant. Also, the reproduction number of the model was accurately worked out. The stability of the model showed that the disease free and endemic equilibrium is stable if necessary conditions are satisfied. The numerical simulations graphically showed the effect of the control strategies. The work is well organized and suitable for indexing.\nHowever, a few issues should be addressed:\nThe manuscript needs to be properly arranged as some of the matrices are too big. The boxes should be adjusted. Under stability analysis of the DFE, the R0 is not clearly shown. Since conclusion depends on the value of R0, it is necessary that R0 is properly substituted in the equation. In the sensitivity section, the sensitivity value need to be presented in order to know how sensitive each parameters are. The + and - signs are not enough Graphs are not bold enough. Revisit the conclusion under stability of DFE to drive home your findings In figure 5, I suggest that more than two values of  ζ  be used. In the conclusion, the novelty of the work is not elaborately discussed. Please rewrite this part to show what the contribution to knowledge is. There should be a paragraph discussing earlier work on diseases such as Ebola, covid-19 and the likes threatening global health. Such articles like, but not limited to 'qualitative analysis and dynamical behaviour of a Lassa haemorrhagic fever model with exposed rodents and saturated incidence rate' would help.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "158041",
"date": "05 Jan 2023",
"name": "Fatmawati Fatmawati",
"expertise": [
"Reviewer Expertise Mathematical modeling in life science",
"optimal control",
"fractional modelling."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have read this article. Some of my critical comments include:\nPlease explore the novelty of this article. What is the difference between this article and previous research that already exists.\n\nI don't see the connection between the existence of the endemic equilibrium point and basic reproduction number (R0). Likewise to prove the stability of the disease-free equilibrium. The authors only mention that if R0 < 1 then the disease-free equilibrium is LAS, while depending on R0 cannot be proven exactly\n\nPlease also check for proof of global stability of the disease-free equilibrium.\n\nThe authors must proofread very carefully the language of the manuscript.\n\nCarefully check in whole manuscript, dot, and comma after each equation.\n\nThen numerical results need to be discussed in more details.\n\nThe conclusion sections should highlight the main findings of this study.\n\nReferences should be up to date and must follow the style correctly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "159237",
"date": "25 Jan 2023",
"name": "Ropo Ebenzer Ogunsakin",
"expertise": [
"Reviewer Expertise Statistician",
"Biological and Computational Mathematics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors developed a new mathematical model for the transmission dynamics of HIV and AIDS and the model was rigorously analysed. The authors considered the impact of three major strategies (impact of awareness, testing and follow up) which are important in controlling the transmission dynamics of HIV/AIDS. The six- compartmental model of HIV/AID presented are: Susceptible, (S), representing people who prone to be infected with HIV in engage in risk habits/factors, those who are unaware of their HIV status (HU), those who become aware after testing (HA), those who are placed on treatment after becoming aware (HT); the AIDS individuals (AA) due to non-adherence to treatment and the AIDS on treatment population (AT). The author showed that the model is positive through a given region in their analyses and the reproduction number of the model was correctly worked out. The effects of the strategies were graphically represented in the numerical simulations. The title is peaks a volume to the context of the article.\nThe work is well organized and approved for indexing. I have the following observation/comment on the paper:\nThe overall command of English looks good, however there are some grammatical errors in the paper. Also the authors should check for incomplete sentences in the paper. Some representation can be made to reduce the size of the matrices as they are big or boxes should be adjusted. It is necessary that the biological meaning of R0 be properly stated. In the sensitivity section, the authors can as well placed the real values of the indices. The conclusion looks good, the novelty of the work is discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/11-1145
|
https://f1000research.com/articles/12-225/v1
|
28 Feb 23
|
{
"type": "Research Article",
"title": "Examination of Lifetime Established Use Criteria for Adult Tobacco Product Usersâ¯",
"authors": [
"Lai Wei",
"Mohamadi Sarkar",
"Thaddaeus Hannel",
"Edward Largo",
"Raheema Muhammad-Kah",
"Mohamadi Sarkar",
"Thaddaeus Hannel",
"Edward Largo",
"Raheema Muhammad-Kah"
],
"abstract": "Background: Lifetime established use criteria are essential to distinguish tobacco product experimental and established users; however, these criteria are not as well defined for some tobacco product categories. The objective of this analysis is to examine various lifetime use criteria to facilitate an objective characterization of experimental versus established use behaviors. Methods: Our analyses were conducted using the Population Assessment of Tobacco and Health (PATH) Study Waves 1 to 5 datasets. We first examined the level of agreement between non-numerical (having smoked/used the product fairly regularly) and numerical (Â having smoked/used the product 20/50/100 times (e.g., use occasions) or product units) lifetime established use criteria among ever-, current-, and former-tobacco product users of various tobacco products. Logistic regression models were constructed to compare demographics, socioeconomic status, and tobacco product use patterns among current experimental versus current established users. Longitudinal analyses were performed to investigate the association between numerical or non-numerical criteria and discontinuation of past 30-day use at follow-up waves. Results: For tobacco product categories other than cigarette, we identified the following numerical thresholds as the objective characterization of established use: 50 for cigars and pipe products and 20 times for hookah, electronic nicotine products, smokeless, snus, and dissolvable tobacco products, based on level of agreement with the non-numerical criterion (kappa coefficients ranging from 0.3 to 0.6) and also with consideration of existing consensus. Significant differences were observed in demographics, socioeconomic status, tobacco product use and discontinuation of past 30-day use patterns when comparing current experimental and current established users with either non-numerical or numerical criteria. Conclusions: This study is the first PATH data analysis to derive quantitative thresholds of lifetime established use criteria to characterize experimental versus established use behaviors. Consistent applications of the lifetime established use criteria in data analysis and reporting would improve harmonization in research findings.",
"keywords": [
"Tobacco established use behavior",
"experimental use behavior",
"lifetime established use thresholds",
"numerical lifetime established use criterion",
"non-numerical lifetime established use criterion."
],
"content": "Introduction\n\nDistinguishing experimental and established tobacco product users is essential to study product use behavior (e.g., consumption or transition patterns), determine product adoption/cessation versus trial, and compare perceptions and intentions measures. The use of lifetime consumption of 100 cigarettes threshold has been well accepted to define established cigarette smokers.1â3 However, the lifetime established use criteria for other combustible or smoke-free product categories are less clear. Sánchez-Romero et al. conducted a comparison of tobacco product prevalence by different frequency of use thresholds and demonstrated lower variability in cigarette smoking prevalence by different current use frequency thresholds, which may be due, in part, to the adoption of the â100 cigarette lifetimeâ criteria for prevalence estimation.4\n\nThere have been various efforts made to define established use for tobacco categories other than cigarette. For example, âever smoked 50 or more cigarsâ has been adopted as the lifetime established use criterion for cigars from national surveys, such as the National Health Interview Survey (NHIS) and the National Adult Tobacco Survey (NATS).5â8 âEver use of smokeless tobacco greater than or equal to 20 timesâ has been applied as the established use criterion for smokeless tobacco products from national surveys, such as the NHIS.9â11 However, there is a lack of consensus regarding the numerical criteria of lifetime established use for electronic nicotine delivery system (ENDS) or other tobacco products. As examples, in the 2012â2013 and 2013â2014 NATS, numerical threshold of â1 timeâ (i.e., ever use) was used as threshold for lifetime use for hookah, e-cigarette, snus, and dissolvable tobacco products while the thresholds for conventional products were 100 for cigarettes, 50 times for cigars and pipe products and 20 times for chew, snuff, or dip.12,13 On the other hand, the thresholds were 50 times for a pipe tobacco product in the 2000 NHIS and 2005 NHIS surveys,8 and 50 times for e-cigarettes.14â17 Lastly, âfairly regularlyâ has been used in national surveys as a way to describe a persistent and stable product usage state. There are survey questions such as âsmoke cigarettes fairly regularly throughout the dayâ (National Survey on Drug Use and Health (NSDUH)) (with response options of 1-Not at all true to 5-Extremely true), and âhow old (were/was) (you/name) when (you/he/she) first started smoking cigarettes fairly regularlyâ (Tobacco Use Supplement - Current Population Survey (TUS-CPS)). The lifetime criterion of âhaving ever used the tobacco product fairly regularlyâ has been widely used now for tobacco products other than cigarettes (e.g., electronic nicotine products, smokeless tobacco, and snus products) with the inclusion of the questions in the Population Assessment of Tobacco and Health (PATH) Study.2,7,11,18â25\n\nThe PATH study is an on-going nationally representative, longitudinal cohort study of U.S. civilian non-institutionalized youth and adults. The study was launched in 2011 as a collaborative effort by the U.S. Food and Drug Administration (FDA), Center for Tobacco Products (CTP), the National Institutes of Health (NIH), and the National Institute on Drug Abuse (NIDA). The first wave of data from the PATH study was collected in 2013, and seven waves have been implemented or planned through 2022. The study generates longitudinal data on tobacco use behaviors, including patterns of use, attitudes, beliefs, exposures, and health consequences associated with the use of tobacco products. The PATH study interviews adult tobacco users and non-users about the use of multiple tobacco products, including cigarettes, e-cigarettes and electronic nicotine products, cigars (traditional cigars, cigarillos, filtered cigars), smokeless tobacco, snus pouches, pipe tobacco, hookah, and dissolvable tobacco products. There are two types of lifetime established use criteria that can be derived from PATH study questionnaires: (1) non-numerical criterion of having ever smoked/used the product fairly regularly, and (2) numerical criterion of having smoked/used the product X number of product units or used X or more times (e.g., 100 or more cigarettes, 100 or more times). Our secondary analyses provide a comprehensive overview of the response patterns and relationships between these two types of criteria for available tobacco product categories in PATH. To our knowledge, this is the first analysis to examine the differences in characteristics, such as demographics (e.g., age, sex), socioeconomic status (e.g., household income, education), tobacco product use and discontinuation of past 30-day use patterns, between experimental and established users across a wide spectrum of tobacco product categories. Additionally, through our review of the response patterns and relationships, we have provided recommendations of numerical thresholds that are in fair to substantial agreement with the non-numerical thresholds and can be used to facilitate alignment and harmonization in future tobacco research.\n\n\nMethods\n\nOur analyses were based on adult participants from the PATH study who were 18 years and older at each interview wave. At the time of our analyses, there were five waves of publicly available PATH data sets26 with annual data collections between Wave 1 (September 2013 to December 2014) to Wave 4 (December 2016 to January 2018), and biennial collections after Wave 4 (e.g., Wave 5 data collection: December 2018 to November 2019). We derived the lifetime established use criteria based on two lifetime use questions in PATH1: the non-numerical criterion from the survey question âhave you ever smoked/used the tobacco product fairly regularly?â and2 and the numerical criterion based on âhow many [units] have you smoked/used in your entire life?â Both questions were asked to ever users of the tobacco product(s) except for the cigarette category. Only ever smokers who had smoked 100 or more cigarettes were asked if they âhave ever smoked cigarettes fairly regularlyâ. PATH defines established tobacco product users as âhaving smoked 100 or more cigarettesâ for cigarette smokers and âhaving smoked/used the tobacco product fairly regularlyâ for other tobacco product users. These definitions were used to implement survey conditional branching (i.e., skip logic) in the PATH questionnaires, and to assess tobacco use patterns and use histories in secondary data analysis. We did not stratify our analyses by sex as we do not expect the lifetime established use criteria to differ by sex. Sex was taken into account as one covariate with other demographic variables in logistic regression models to investigate the association with reaching lifetime established use criteria for various tobacco products.\n\nDue to the longitudinal nature of the PATH study, our cross-sectional analyses relied mainly on Wave 1 data to compare experimental versus established use for available tobacco product categories. During the data collection period of Wave 1 (2013â2014) to Wave 5 (2018â2019), changes were made to the PATH survey questionnaires pertaining to the assessment and description of some tobacco product categories to accommodate for the evolution of the tobacco products. For instance, âe-cigarettesâ evaluated in Wave 1 expanded to include âelectronic nicotine productsâ such as e-cigarettes, e-hookahs, e-cigars, e-pipes, personal vaporizers, vape pens, and hookah pens in Waves 2 to 5. Accordingly, there were also changes in the lifetime established use questions for electronic nicotine products and snus, as summarized in Table 1, to increase consistency in survey questions across tobacco product categories. For example, the unit for electronic nicotine product category was âdisposable e-cigarettes or e-cigarette cartridgesâ in Wave 1, âe-cigarettes or total milliliters (if with e-liquid)â in Wave 2 and âtimesâ used in Waves 3 to 5.\n\n* The primary electronic nicotine product could be an e-cigarette, e-cigar, e-pipe, e-hookah, or other electronic nicotine product based on the question âWhich electronic nicotine product do you use most often?â in Wave 3.\n\nâ Dissolvable tobacco product is described in PATH Wave 1 as âYou donât smoke dissolvable tobacco products â they are made of finely ground flavored tobacco that dissolves in your mouth. Dissolvable tobacco products come in a variety of shapes, including small round pellets, thin sticks, and flat strips. Some common brands are Ariva, Stonewall, and Camel Orbs, Sticks or Strips.â\n\nFor each tobacco product category, we first presented demographics, socioeconomic status, and tobacco product use patterns among current users, current experimental users, and current established users based on the PATH definition of established use. Table 2 lists the descriptive statistics generated for sex, age, race/ethnicity, household income, education, current use status (every day, and some days), and concurrent use status (exclusive, and dual/poly use). The definition of exclusive use is reported as current (âevery dayâ or âsome daysâ) use of the tobacco product and no current use of any other tobacco products. The definition of dual/poly use is current use of two or more tobacco products among the tobacco product categories being assessed.\n\nâ Caution: low sample size. The estimates may not be reliable due to low base sample size (n< 50) or coefficient of variation greater than 30.\n\nTo evaluate the two sets of lifetime established use definitions, we presented the response patterns among current users in Figure 1. We then examined the level of agreement between the two sets of responses using kappa coefficient among ever, current, and former tobacco product users for all available tobacco categories except for cigarette (Table 3). Ever tobacco use is defined as having ever used the product, even once. Current tobacco use is defined as currently using the tobacco product âevery dayâ or âsome daysâ. Former tobacco use is defined as having ever used the tobacco product and reported using the tobacco product ânot at allâ now.\n\nCross-sectional analyses were performed based on PATH Wave 1 data to study the response pattern of lifetime usage based on numerical and non-numerical criterion among current tobacco product users for each tobacco category.\n\nSample sizes of current tobacco user groups: N = 13,470 (cigarette), 2,575 (cigar-traditional), 2,948 (cigar-cigarillo), 1,290 (cigar-filtered), 703 (pipe), 3,040 (hookah), 3,586 (electronic nicotine products), 1,867 (smokeless), 480 (snus), and 63 (dissolvable).\n\nNote: Missing responses of the numerical and non-numerical criteria are excluded from the analysis.\n\n*The fairly regular use question was only assessed among those who had smoked 100 or more cigarettes for current cigarette smokers.\n\nKappa Agreement\n\n0.01-0.20âââSlight\n\n0.21-0.40âââFair\n\n0.41-0.60âââModerate\n\n0.61-0.80âââSubstantial\n\n0.81-0.99âââAlmost perfect\n\nCross-sectional analyses were performed based on PATH Wave 1 data to study the levels of agreement between non-numerical criteria (i.e., having used the product fairly regularly), and numerical criteria (i.e., having used the product 20/50/100 or more units) among ever-, current-, and former tobacco-users.\n\nWave 1 Tobacco User Group Sample size:\n\nCigarette: ever users (n=25,183), current users (n=13,478), former users (n=11,571).\n\nCigar - Traditional: ever users (n=10,220), current users (n=2,581), former users (n=7,624).\n\nCigar - Cigarillo: ever users (n=13,701), current users (n=2,964), former users (n=10,565).\n\nCigar â Filtered: ever users (n=6,636), current users (n=1,294), former users (n=5,323).\n\nPipe: ever users (n=7,178), current users (n=708), former users (n=6,460).\n\nHookah: ever users (n=10,623), current users (n=3,047), former users (n=7,563).\n\nElectronic nicotine products: ever users (n=11,523), current users (n=3,640), former users (n=7,866).\n\nSmokeless: ever users (n=6,611), current users (n=1,870), former users (n=4,538).\n\nSnus: ever users (n=2,917), current users (n=478), former users (n=2,436).\n\nDissolvable: ever users (n=347), current users (n=67), former users (n=277).\n\n* The fairly regular use question was only assessed among those who had smoked 100 or more cigarettes for current cigarette smokers, so we are not able to examine the level of agreement.\n\nSuggested numerical thresholds were discussed (Table 4) based on the level of agreement with the non-numerical fairly regular use criterion and review of existing lifetime usage criteria from national surveys or literature. With suggested numerical thresholds, logistic regression models were fitted to evaluate the covariates of reaching non-numerical and numerical lifetime established use criterion (Table 5). The reference group consisted of experimental users in each tobacco product category. Covariates in the logistic regression models included self-report sex, age, race/ethnicity, household income, education, current use, and concurrent use status.\n\n* One time refers to a typical session when the participant picks up the product to use it. For example, description in PATH Wave 5 questionnaire is âthe time the participant picks up the ENDS product to use it. Multiple puffs can be taken within one session.â\n\n* p-value < 0.05\n\nPrior research has demonstrated that established tobacco product users are less likely to discontinue past 30-day use.16,27 In this article, we examined the predictive validity of the suggested numerical thresholds by constructing generalized estimating equation (GEE) logistic regression models among current tobacco product users at baseline wave (Table 6). These models allow an understanding of the association between established use and discontinuation of past-30 day use at follow-up waves (i.e., in a one-year interval) based on Wave 1 to Wave 4 data. Follow-up wave means the wave after the baseline wave. Baseline waves include Wave 1, 2 and 3. Our analyses only included the first four waves because there was a one-year interval between Wave 1 to Wave 2, Wave 2 to Wave 3, and Wave 3 to Wave 4. There was a two-year interval between Wave 4 and Wave 5. Therefore, Wave 5 data was not included in the GEE logistic regression models.\n\n* p-value < 0.05.\n\nCovariates in GEE logistic regression models include self-report sex, age, race/ethnicity, household income, education, current use status, concurrent use status, non-numerical lifetime established use criterion, and numerical lifetime established use criterion. Due to sample size constraints, models were only constructed for cigarette, traditional cigar, cigarillo, electronic nicotine product, and smokeless tobacco product categories.\n\nAll statistical analyses were conducted in SAS 9.4. The appropriate cross-sectional and longitudinal weights and replicate weights were used with balanced repeated replication (BRR) using a Fayâs adjustment value of 0.3 for variance estimation as required in the PATH user guide.26 For proportions, confidence intervals were computed using the modified Wilson score option. The GEE logistic regression models were constructed using longitudinal weights and replicate weights21 to account for the complex survey design.\n\nTo account for survey questionnaire changes between waves, additional cross-sectional analyses were conducted based on Waves 1 to 5 data sets for the electronic nicotine product and snus product categories. We presented demographics, socioeconomic status, and tobacco product use patterns for current, current experimental, and current established e-vapor users based on Waves 1 to 5 data (Table 7). We showed the lifetime established use response patterns (Figure 2) and level of agreement coefficients for electronic nicotine products (Table 8) to investigate the dynamic landscape of this emerging tobacco category. Level of agreement coefficients were also generated for snus products (Table 9) using five waves of data as the unit of the numerical threshold changed from number of pouches in Waves 1â3, to number of times in Waves 4 and 5 (see Table 1 for details).\n\n⌠PATH data collection period: September 2013 to December 2014 (Wave 1), October 2014 to October 2015 (Wave 2), October 2015 to October 2016 (Wave 3), December 2016 to January 2018 (Wave 4), and December 2018 to November 2019 (Wave 5).\n\nCross-sectional analyses of PATH Wave 1 to Wave 5 data sets were performed.\n\nPATH data collection period: September 2013 to December 2014 (Wave 1), October 2014 to October 2015 (Wave 2), October 2015 to October 2016 (Wave 3), December 2016 to January 2018 (Wave 4), and December 2018 to November 2019 (Wave 5).\n\nSample sizes of current electronic nicotine product user group: n = 3,586 (Wave 1), 3,041 (Wave 2), 2,618 (Wave 3), 2,888 (Wave 4), and 3,947 (Wave 5).\n\nNote: Missing responses of the numerical and non-numerical criteria are excluded from the analysis.\n\nRespondents were only asked the numerical lifetime usage question if they had not smoked/used the tobacco product 100 or more units at prior wave. Therefore, the status of having smoked/used 100 or more units was imputed based on responses at current wave and prior waves.\n\nKappa Agreement\n\n0.01-0.20âââSlight\n\n0.21-0.40âââFair\n\n0.41-0.60âââModerate\n\n0.61-0.80âââSubstantial\n\n0.81-0.99âââAlmost Perfect\n\nCross-sectional analyses were performed based on PATH Waves 1 to 5 data sets. Non-numerical criteria is defined as âHaving Used the Product Fairly Regularlyâ. Numerical Criteria is defined as âHaving Used the Product 20/50/100 Or More Unitsâ.\n\nSample sizes:\n\nWave 1: electronic nicotine products ever users (n=11,523), current users (n=3,640), former users (n=7,866).\n\nWave 2: electronic nicotine products ever users (n=11,275), current users (n=3,648), former users (n=6,671).\n\nWave 3: electronic nicotine products ever users (n=13,335), current users (n=2,671), former users (n=7,351).\n\nWave 4: electronic nicotine products ever users (n=15,969), current users (n=2,958), former users (n=10,382).\n\nWave 5: electronic nicotine products ever users (n=16,478), current users (n=4,184), former users (n=10,260).\n\n⌠PATH data collection period: September 2013 to December 2014 (Wave 1), October 2014 to October 2015 (Wave 2), October 2015 to October 2016 (Wave 3), December 2016 to January 2018 (Wave 4), and December 2018 to November 2019 (Wave 5).\n\nKappa Agreement\n\n0.01-0.20âââSlight\n\n0.21-0.40âââFair\n\n0.41-0.60âââModerate\n\n0.61-0.80âââSubstantial\n\n0.81-0.99âââAlmost Perfect\n\nSample sizes:\n\nWave 1: snus ever users (n=2,917), current users (n=478), former users (n=2,436).\n\nWave 2: snus ever users (n=2,798), current users (n=299), former users (n=2,364).\n\nWave 3: snus ever users (n=2,857), current users (n=288), former users (n=1,968).\n\nWave 4: snus ever users (n=3,641), current users (n=453), former users (n=2,715).\n\nWave 5: snus ever users (n=3,465), current users (n=355), former users (n=2,692).\n\n⌠PATH data collection period: September 2013 to December 2014 (Wave 1), October 2014 to October 2015 (Wave 2), October 2015 to October 2016 (Wave 3), December 2016 to January 2018 (Wave 4), and December 2018 to November 2019 (Wave 5).\n\n\nResults\n\nTable 2 shows the distinct differences between experimental and established users in terms of demographics, socioeconomic status, and tobacco use patterns among current-, current experimental-, and current established-users based on the PATH definition of established use (i.e., 100+ for cigarettes, and fairly regular use for all other tobacco product categories). Firstly, we did not observe differences in sex distributions between experimental versus established users in most of the tobacco categories. The proportions of male users were relatively higher in established users compared to experimental users for smokeless and snus products, and lower for dissolvable tobacco products. Secondly, there seemed to be notable differences in the age distributions of experimental versus established users. The proportion of young adults (i.e., 18â24 years of age) was higher among experimental cigarette smokers compared to established smokers (24.1% versus 14.1%). Similar differences were observed in filtered cigars, pipe, hookah, smokeless, and dissolvable tobacco products. The impact of the lifetime use criterion on age distribution of experimental versus established tobacco product users was investigated further using logistic regression models and described in the ensuing sections of this manuscript.\n\nIn general, the proportions of everyday use were higher among established users when compared to experimental users. We observed predominant proportions of dual/poly use among various tobacco product user groups except for current cigarette smokers. Compared to established users, the proportions of dual/poly use were higher among experimental users for the majority of tobacco product groups including cigarillos, filter cigars, pipe, electronic nicotine products, smokeless, snus, and dissolvable tobacco products. Table 7 depicts the changes in demographics, socioeconomic status, and tobacco use patterns among Wave 1 to Wave 5 current electronic nicotine product users. We observed an increasing proportion of young adults (i.e., 18â24 years of age) (20.9% in Wave 1 to 34.8% in Wave 5) among current electronic nicotine product users. Furthermore, every day electronic nicotine product use increased from 21.3% in Wave 1 to 40.1% in Wave 5, and exclusive electronic nicotine product use increased from 13.7% in Wave 1 to 39.6% in Wave 5.\n\nResponse patterns of lifetime usage vary greatly among current users of each tobacco product category (Figure 1). The five levels of numerical lifetime use range were color coded for depiction as follows: 1â10 units (orange), 11â20 units (green), 21â50 units (blue), 51â99 units (yellow) and 100+ units (red). Using the same color schemes, but differing degrees of color intensity helped distinguish between reported ânot fairly regular useâ (lighter colors) and âfairly regular useâ (darker colors). We observed that over 80% of current cigarette smokers have smoked 100 or more cigarettes and among those, a majority reported having smoked fairly regularly. For other categories, while the proportions of âfairly regular useâ (darker color) tended to be higher in the range of 51â99 units or 100+ units, the reported âfairly regular useâ occupied a notable proportion in all five levels of the numerical lifetime usage ranges. When looking at electronic nicotine product lifetime use patterns in Wave 1, more than 60% of current electronic nicotine product users only used the products 1-10 times and about one third of them reported that they had used âfairly regularlyâ. The proportion that reported fairly regular use within each numerical lifetime usage level for electronic nicotine products seemed relatively higher when compared to other tobacco product categories. Given the continuous evolution of electronic nicotine products, the proportions of having used 20 or more units increased from about 18% in Wave 1 (i.e., 20 or more disposable e-cigarettes or e-cigarette cartridges) to about 78% in Wave 5 (20 or more times) (Figure 2).\n\nTable 3 captures the level of agreement between the non-numerical (i.e., fairly regular use) criterion and numerical criteria with three different thresholds (i.e., 20 units, 50 units, and 100 units) among ever, current, and former users of respective tobacco products. We summarized our observations as follows:\n\n- Cigarette: The level of agreement could not be assessed for cigarette category as only cigarette smokers who have smoked 100 or more cigarettes were asked the fairly regular use question.\n\n- Cigar: In general, observations pointed to moderate agreements (with kappa ranges from 0.38 to 0.50) among ever, current, and former cigar smokers when the threshold reached 50 or more traditional cigars/cigarillos/filtered cigars.\n\n- Pipe: Moderate to substantial agreements (kappa ranges from 0.46 to 0.63) were evident from the 50 or more bowls threshold with ever, current, and former users.\n\n- Hookah: Moderate agreements (kappa ranges from 0.48 to 0.55) were seen with the 20 or more times for ever, current, and former users. The level of agreement became lower with the threshold raised to 50/100 or more times.\n\n- Electronic Nicotine Products: In Wave 1, observations exhibited only fair agreement when the threshold was 20 or more e-cigarettes. However, as shown in Table 8, in later waves, the level of agreement with fairly regular use became higher with different thresholds. A trend began to emerge of moderate agreement with the threshold of 20 or more e-cigarettes/times among ever and current users from Wave 2. Since Wave 3, moderate agreements were displayed with almost all three thresholds pertaining to ever, current, and former electronic nicotine product users. By Wave 5, moderate to substantial agreement (kappa ranges from 0.46 to 0.62) with fairly regular use was shown with the threshold from 20 or more times among ever, current, and former users of electronic nicotine products. We also observed that increasing the lifetime established use threshold to 50 or 100 or more times was not associated with increased level of agreement for electronic nicotine products.\n\n- Smokeless Tobacco Product: Substantial agreements were observed starting from the threshold of 20 or more times among ever and former smokeless tobacco users (kappa=0.75 and 0.69, respectively). Among current smokeless tobacco users, moderate agreement was displayed with three different thresholds (kappa ranges from 0.56 to 0.60).\n\n- Snus: Similar to the smokeless tobacco products, Wave 1 data suggested moderate to substantial agreement with a threshold of 20 or more among ever and former snus users (kappa=0.61 and 0.60, respectively). Among Wave 1 current snus users, moderate agreement corresponded to thresholds of 50 or 100 or more snus pouches (kappa=0.51 and 0.49, respectively). Additionally, assessment of Wave 1 through Wave 5 data showed (Table 9) moderate agreement was reached with a threshold of 20 or more among snus ever and former users in all five waves. Among snus current users, fair to moderate agreement was observed with all three thresholds.\n\n- Dissolvable: As a novel tobacco product category, fair to moderate agreement was seen with the threshold of 20 or more pieces of dissolvable tobacco products among ever and former users (kappa=0.34 and 0.44, respectively). Among current users, only slight agreement with the thresholds was shown with all three thresholds (kappa ranges from 0 to 0.03). The lifetime established use questions for the dissolvable tobacco product category were only available in Waves 1 and 2 so we were not able to examine the established use behavior further for this category.\n\nWe summarized the suggested numerical lifetime established use criteria in Table 4, by synthesizing our findings with existing survey measures, and definitions that had been previously adopted in publications. These thresholds for suggested numerical lifetime established use were used to compare with the fairly regular use criteria when conducting the logistic regression models in later sections.\n\nAs distinct differences in descriptive statistics were uncovered for demographics, socioeconomic status, and tobacco product use patterns (Table 2), we further examined the correlates of reaching lifetime established use criterion in Wave 1 using logistic regression models (Table 5). In general, we observed female respondents have lower odds of reaching the respective lifetime established use criterion compared to male respondents for most of the tobacco product categories except for cigarettes and pipe tobacco products. Age was a significant covariate for reaching the respective lifetime established use criterion, while in some cases, there were greater associations between age and being established users with the numerical criterion than with non-numerical criterion. For example, among current traditional cigar smokers, compared to the 18â24 age group, older adults were more likely to report having smoked 50 or more cigars in their entire life (25â44 age group, adjusted odds ratio (aOR)=1.5, 95%CI 1.1â1.9; 45+ age group, aOR=3.4, 95%CI 2.7â4.3). On the contrary, no significant differences were found when comparing the odds ratio (OR) of reaching the fairly regular use criterion between younger and older age groups among traditional cigar smokers (25â44 age group, aOR=0.8, 95%CI 0.7â1.0; 45+ age group, aOR=0.8, 95%CI (0.6â1.0).\n\nFor other demographic and socioeconomic variables, the patterns varied across different tobacco product user groups when comparing the numerical versus the non-numerical lifetime established use criterion. For instance, among current electronic nicotine product users, education was a significant covariate of reporting having used fairly regularly but was not a significant covariate of reporting having ever used 20 or more e-cigarettes. Across all tobacco product user groups, consistent results showed that âevery dayâ tobacco product users were much more likely to report reaching either non-numerical or numerical lifetime established use criterion in comparison with âsome dayâ use.\n\nLastly, in regard to dual/poly use, there were some distinct patterns. While the odds of reaching lifetime established use criterion given dual/poly use compared to exclusive use were not significant for most of the combustible tobacco products, there were higher odds of being established users among traditional cigar users (aOR=1.3, 95%CI 1.0â1.6 with lifetime use criterion of 50 or more traditional cigars), and hookah users (aOR=1.3, 95%CI 1.1â1.6 with lifetime use criterion of 20 or more times). For smoke-free tobacco products, there were lower odds of being established users given dual/poly use compared to exclusive use among electronic nicotine product users (aOR=0.7, 95%CI 0.5â1.0 with the fairly regular use criterion, and aOR=0.7, 95%CI 0.5â1.0 with lifetime use criterion of 20 or more disposable e-cigarettes or e-cigarette cartridges (from Wave 1 measure, see Table 1 for reference) and smokeless tobacco product users (aOR=0.5, 95%CI 0.3â0.9 with lifetime use criterion of 20 or more times).\n\nIn general, after adjusting for sex, age, race/ethnicity, household income, education, current and concurrent use status, established tobacco product users (defined using either the numerical or non-numerical lifetime use criterion) were less likely to discontinue past-30 day use at follow-up wave (i.e., in one-year intervals) (Table 6). Similar aORs were displayed with numerical or non-numerical criteria for most tobacco product categories. For example, for electronic nicotine product users, the aOR of discontinuing past 30-day use was aOR=0.6, 95%CI 0.5â0.7 with non-numerical criterion reached, and aOR=0.7, 95%CI 0.6â0.9 with numerical criterion reached. Compared to âsome dayâ users, âevery dayâ users were much less likely to discontinue past 30-day use (cigarette: aOR = 0.2, 95%CI 0.2â0.3; traditional cigar: aOR=0.6, 95%CI 0.4â0.9; cigarillo: aOR=0.6 95%CI 0.5â0.8; electronic nicotine product: aOR=0.4 95%CI 0.3â0.4; and smokeless tobacco: aOR=0.2 95%CI 0.2â0.3). Dual/poly use was not statistically associated with discontinuation of past-30 day use for cigarette, traditional cigar, and cigarillo categories. However, significant associations were shown among electronic nicotine and smokeless tobacco product users. Compared to exclusive users, dual/poly users were more likely to discontinue past 30-day use of the respective products (electronic nicotine product: aOR=1.6 95%1.3â2.0; smokeless tobacco: aOR=1.8 95%CI 1.4â2.4).\n\n\nDiscussion\n\nAlthough the origin and use of 100 or more cigarettes as lifetime established use criterion may be somewhat arbitrary, it has become an important screener for ânever regularâ cigarette smoking among adults. In our analyses, we demonstrate the importance of distinguishing between experimental and established use for available tobacco product categories in PATH. For example, adult tobacco users classified as experimental users are more likely to be younger, use on âsome daysâ (versus âevery dayâ), and are less likely to have continued tobacco use at follow-up compared to those classified as established users. Our analyses also reveal that the criterion of âfairly regularlyâ can be broadly interpreted by adult tobacco users, and thus, suggest that numerical criteria may provide a more objective characterization of established users. That said, subjective criteria may still be warranted to characterize users of new products until more is known about the use behavior with these products. Additionally, it is important to adopt the phrase âestablished useâ when a predefined established use criterion has been applied. For example, for cigarette smokers, we could differentiate âever established useâ with âever useâ based on âhaving smoked 100 or more cigarettesâ. Some national surveys (e.g., NHIS, the Behavioral Risk Factor Surveillance System (BRFSS)) only ask cigarette smoking related questions if the respondent reported having ever smoked 100 or more cigarettes. With this conditional branching, it is not possible to evaluate the true âever smokerâ population from these national surveys.\n\nOne key limitation of this study is that our findings are based on analysis of existing PATH survey questions with self-reported data. For cigarette smokers, the concept of âfairly regularâ was only asked of those whose threshold reached 100+ cigarettes, which does not allow us to further investigate the âfairly regularâ use among experimental smokers. Additionally, dissolvable tobacco products might have been on the U.S. market during early waves of PATH data collection (i.e., 2013â2015), but a majority of the products were no longer sold in the U.S. Moreover, when studying use behavior and discontinuation of past 30-day use patterns, the evolution of new tobacco product categories necessitating changes implemented in survey questionnaires makes comparison across waves challenging. Given the longitudinal nature of the PATH study, some cross-sectional analysis comparisons with five waves of data should be interpreted with caution as the responses may come from the same individuals when they were followed up at later waves. Lastly, we were not able to assess certain emerging tobacco product categories such as heated tobacco products or tobacco-free oral nicotine products, as these products were not included in the PATH surveys. It is worth noting that some existing research has utilized a threshold of â100 or more heatsticksâ to identify established IQOS users.28 Additionally, due to the similarity between tobacco-free oral nicotine products and snus, a lifetime use of â20 or more timesâ may be a relevant threshold for established oral nicotine product use.\n\nIn addition to lifetime ever use criteria discussed in this article, there have been other lifetime established use measures/definitions used in national surveys or recent publications. For example, some surveys ask questions such as âhas there ever been a period in your life when you smoked cigarettes every day for at least 30 days?â (NSDUH), and âfor how long have you smoked every day?â (TUS-CPS). Current use frequency (e.g., used 1+, 5+, 10+, 20+, and 30 days in the past 30 days) has also been adopted by other studies to define established use.4,24,27,29 As the key objective of our research is to study numerical lifetime established use criterion, we did not look into other questions in PATH to study established use definitions based on current use frequency or other use patterns.\n\nOur analysis is the first PATH secondary data analysis to derive quantitative thresholds of lifetime established use criteria to better characterize experimental versus established use behaviors. We provided recommendations of numerical lifetime established use criteria (Table 4) by synthesizing our findings with existing survey measures, and definitions that had been previously adopted in publications. Our analyses showed that, when studying emerging tobacco product categories (e.g., electronic nicotine products), the proportion of established use may be higher with the non-numerical fairly regular use criterion compared to numerical criterion in the early stages (i.e., Waves 1 and 2). But as the product becomes more prevalent, the numerical criterion serves as a more objective alternative to differentiate experimental users from established users. While the selection of the numerical thresholds may be somewhat arbitrary as the original of 100 or more cigarettes, the selection of â20 timesâ as the numerical lifetime established use threshold for smoke-free tobacco products, including electronic nicotine, smokeless, snus, and dissolvable tobacco products, may improve the consistency in survey design and data analysis. While we did observe some significant differences in demographics (e.g., sex and age) in terms of reaching lifetime established use criteria for some tobacco categories, it may not be desirable to have the lifetime established use criteria differ by demographics. In practice, applying the same criteria is key to facilitating comparisons across sub-populations. As the tobacco landscape rapidly evolves, it becomes increasingly important to develop established use criteria for newer products and re-examine criteria for existing products to characterize product use behavior (e.g., consumption or transition patterns), determine product adoption/cessation versus trial, and compare perceptions and intentions measures.\n\n\nConclusion\n\nThe adoption of lifetime use criteria would facilitate the study of experimental and established tobacco product users as distinct differences have been demonstrated in terms of demographics, socioeconomic status, use, and discontinuation of past 30-day use patterns. As new tobacco products emerge in the market, non-numerical criterion may be a good approach to identify regular users at an early stage. In comparison, the numerical lifetime use criterion provides a more objective characterization of lifetime established use when the new tobacco product becomes more widely used. The suggested numerical lifetime use thresholds should be evaluated further as the tobacco landscape evolves through qualitative and quantitative studies to ensure the thresholds are appropriate to distinguish between experimental and established users. Finally, consistent applications of the lifetime established use criteria in data analysis and reporting would improve harmonization in research findings.\n\nNot applicable. This secondary analysis only used publicly available PATH data sets.\n\n\nAuthor Contributions\n\nLai Wei conceptualized the research design, developed the study group, performed data analysis, and drafted the manuscript. Thaddaeus Hannel and Raheema S. Muhammad-Kah contributed to the research conceptualization, reviewed, and revised the manuscript. Mohamadi Sarkar and Edward Largo reviewed and revised the manuscript. All authors read and approved the final manuscript.",
"appendix": "Data Availability\n\nThe data analyzed are publicly available (https://doi.org/10.3886/ICPSR36498.v13) and can be downloaded from https://www.icpsr.umich.edu/icpsrweb/NAHDAP/studies/36498\n\n\nAcknowledgments\n\nThe authors acknowledge Krushna Agulla for analytical support; Sucharitha Iyer and Andrea Vansickel for reviewing and providing insightful comments on this publication.\n\n\nReferences\n\nBondy SJ, Victor JC, Diemert LM: Origin and Use of the 100 Cigarette Criterion in Tobacco Surveys. Tob. Control. 2009; 18(4): 317â323. PubMed Abstract | Publisher Full Text\n\nKlemperer EM, Hughes JR, Callas PW, et al.: Tobacco and Nicotine Use among Us Adult âNever Smokersâ in Wave 4 (2016â2018) of the Population Assessment of Tobacco and Health Study. Nicotine Tob. Res. 2021; 23(7): 1199â1207. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCornelius ME, Wang TW, Jamal A, et al.: Tobacco Product Use among AdultsâUnited States, 2019. Morb Mortal Weekly Rep. 2020; 69(46): 1736â1742. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSánchez-Romero LM, Cadham CJ, Hirschtick JL, et al.: A Comparison of Tobacco Product Prevalence by Different Frequency of Use Thresholds across Three Us Surveys. BMC Public Health. 2021; 21(1): 1â11. Publisher Full Text\n\nNational Cancer Institute. Cigars: Health Effects and Trends. Tobacco Control Monograph No. 9. Bethesda, MD: U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute. NIH Pub. No. 98-4302, February 1998 .\n\nCorey CG, King BA, Coleman BN, et al.: Little Filtered Cigar, Cigarillo, and Premium Cigar Smoking among AdultsâUnited States, 2012â2013. MMWR Morb. Mortal. Wkly Rep. 2014; 63(30): 650.\n\nCorey CG, Holder-Hayes E, Nguyen AB, et al.: Us Adult Cigar Smoking Patterns, Purchasing Behaviors, and Reasons for Use According to Cigar Type: Findings from the Population Assessment of Tobacco and Health (Path) Study, 2013â2014. Nicotine Tob. Res. 2018; 20(12): 1457â1466. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRostron BL, Corey CG, Gindi RM: Cigar Smoking Prevalence and Morbidity among Us Adults, 2000â2015. Prev. Med. Rep. 2019; 14: 100821. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMazurek JM, Syamlal G, King BA, et al.: Smokeless Tobacco Use among Working AdultsâUnited States, 2005 and 2010. MMWR Morb. Mortal. Wkly Rep. 2014; 63(22): 477â482. PubMed Abstract\n\nRodu B, Plurphanswat N: Mortality among Male Smokers and Smokeless Tobacco Users in the USA. Harm Reduct. J. 2019; 16(1): 1â9. Publisher Full Text\n\nCheng Y-C, Rostron BL, Day HR, et al.: Patterns of Use of Smokeless Tobacco in Us Adults, 2013â2014. Am. J. Public Health. 2017; 107(9): 1508â1514. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohnson SE, Holder-Hayes E, Tessman GK, et al.: Tobacco Product Use among Sexual Minority Adults: Findings from the 2012â 2013 National Adult Tobacco Survey. Am. J. Prev. Med. 2016; 50(4): e91âe100. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgaku IT, King BA, Husten CG, et al.: Tobacco Product Use among AdultsâUnited States, 2012â2013. MMWR Morb. Mortal. Wkly Rep. 2014; 63(25): 542â547. PubMed Abstract\n\nHarrell PT, Simmons VN, Piñeiro B, et al.: E-Cigarettes and Expectancies: Why Do Some Users Keep Smoking? Addiction. 2015; 110(11): 1833â1843. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiovenco DP, Lewis MJ, Delnevo CD: Factors Associated with E-Cigarette Use: A National Population Survey of Current and Former Smokers. Am. J. Prev. Med. 2014; 47(4): 476â480. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBiener L, Hargraves JL: A Longitudinal Study of Electronic Cigarette Use among a Population-Based Sample of Adult Smokers: Association with Smoking Cessation and Motivation to Quit. Nicotine Tob. Res. 2015; 17(2): 127â133. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndrews JA, Hampson SE, Severson HH, et al.: Perceptions and Use of E-Cigarettes across Time among Emerging Adults. Tob. Regul. Sci. 2016; 2(1): 70â81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKasza KA, Ambrose BK, Conway KP, et al.: Tobacco-Product Use by Adults and Youths in the United States in 2013 and 2014. N. Engl. J. Med. 2017; 376(4): 342â353. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLevy DT, Yuan Z, Li Y, et al.: An Examination of the Variation in Estimates of E-Cigarette Prevalence among Us Adults. Int. J. Environ. Res. Public Health. 2019; 16(17): 3164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWei L, Muhammad-Kah RS, Hannel T, et al.: The Impact of Cigarette and E-Cigarette Use History on Transition Patterns: A Longitudinal Analysis of the Population Assessment of Tobacco and Health (Path) Study, 2013â2015. Harm. Reduct. J. 2020; 17(1): 1â12. Publisher Full Text\n\nKasza KA, Edwards KC, Tang Z, et al.: Correlates of Tobacco Product Cessation among Youth and Adults in the USA: Findings from the Path Study Waves 1â3 (2013â2016). Tob. Control. 2020; 29(Suppl 3): s203âs215. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWheldon CW, Kaufman AR, Kasza KA, et al.: Tobacco Use among Adults by Sexual Orientation: Findings from the Population Assessment of Tobacco and Health Study. LGBT health. 2018; 5(1): 33â44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson JN, Wang B, Jackson KJ, et al.: Characteristics of Hookah Tobacco Smoking Sessions and Correlates of Use Frequency among Us Adults: Findings from Wave 1 of the Population Assessment of Tobacco and Health (Path) Study. Nicotine Tob. Res. 2018; 20(6): 731â740. PubMed Abstract | Publisher Full Text\n\nWeaver SR, Kim H, Glasser AM, et al.: Establishing Consensus on Survey Measures for Electronic Nicotine and Non-Nicotine Delivery System Use: Current Challenges and Considerations for Researchers. Addict. Behav. 2018; 79: 203â212. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRodu B, Plurphanswat N: E-Cigarette Use among Us Adults: Population Assessment of Tobacco and Health (Path) Study. Nicotine Tob. Res. 2018; 20(8): 940â948. Publisher Full Text\n\nUnited States Department of Health and Human Services. National Institutes of Health. National Institute on Drug Abuse, and United States Department of Health and Human Services. Food and Drug Administration. Center for Tobacco Products. Population Assessment of Tobacco and Health (PATH) Study [United States] Public-Use Files. Inter-university Consortium for Political and Social Research [distributor], 2021-09-29. Publisher Full Text\n\nBrouwer AF, Levy DT, Jeon J, et al.: The Impact of Current Tobacco Product Use Definitions on Estimates of Transitions between Cigarette and Ends Use. Nicotine Tob. Res. 2022; 24: 1756â1762. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAfolalu EF, Langer P, Fischer K, et al.: Prevalence and Patterns of Tobacco and/or Nicotine Product Use in Japan (2017) after the Launch of a Heated Tobacco Product (Iqos®): A Cross-Sectional Study. F1000Res. 2021; 10(504): 504. Publisher Full Text\n\nAmato MS, Boyle RG, Levy D: How to Define E-Cigarette Prevalence? Finding Clues in the Use Frequency Distribution. Tob. Control. 2016; 25(e1): e24âe29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgaku IT, Awopegba AJ, Filippidis FT: The Impact of Inter-Survey Differences in the Definition of Current Smokeless Tobacco Use on Comparability of Us National and State-Specific Prevalence Estimates, 2009â2011. Prev. Med. 2015; 74: 86â92. PubMed Abstract | Publisher Full Text\n\nBiener L, Roman AM, Mc Inerney SA, et al.: Snus Use and Rejection in the USA. Tob. Control. 2016; 25(4): 386â392. Publisher Full Text"
}
|
[
{
"id": "246306",
"date": "07 May 2024",
"name": "Bontle Mbongwe",
"expertise": [
"Reviewer Expertise Public Health",
"toxicology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is the first  to assess lifetime tobacco product use criteria to facilitate a realistic characterization of experimental versus established tobacco product use behaviours. The study proposes new numerical life-time use thresh-holds  for various tobacco product use. The researchers emphasize the importance of this research in light  of the changing tobacco control landscape with emerging new tobacco product categories such as electronic nicotine products and the existing non-numerical and numerical lifetime use criteria.  Of note is that the proposed thresholds for tobacco product categories (whilst subject to validation through further research) could assist  in improving the harmonisation of research findings.\nThe purpose of the publication is well articulated in the abstract and background information of the article. Whilst the results  of this work are well written and explained  generally in the results section, the  abstract may need some minor improvements to represent and summarize the article's major points.\nAbstract Specify the  significant differences observed. Specify the take-home message in the conclusion.\nResults: Be consistent  in reporting clear aOR and confidence intervals in all relevant sections. Some sections report significant/non significance covariates yet not specified.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-225
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https://f1000research.com/articles/12-220/v1
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27 Feb 23
|
{
"type": "Research Article",
"title": "The impact of COVID-19 severity on pregnancy outcomes among Iraqi women: a retrospective observational study",
"authors": [
"Othman Sami Salih",
"Manal Khalid Abdulridha",
"Manal Khalid Abdulridha"
],
"abstract": "Background: COVID-19-infected pregnant women may be asymptomatic or symptomatic. Symptomatic tend to have a severe infection and could be at increased risk of complications such as preterm birth, NICU, and stillbirth. This study aimed to identify the most common signs and symptoms and assess the pre-existing risk of COVID-19 infection during pregnancy: length of hospital stay, premature labor, and complications according to the severity of COVID-19 illness. Methods: This retrospective study was conducted in the obstetric and gynecological wards of 359 pregnant women. After reviewing medical records, patient and disease data were collected. Results: Most patients were asymptomatic and mild. The most common signs and symptoms were fever, cough, and dyspnea. COVID-19 infected pregnant with a medical history of hypertension, or diabetes had more severe COVID-19 infection. COVID-19-infected pregnant women with severe illness or aged ⥠40 years had a prolonged hospital stay. Severe COVID-19 disease was significantly associated with preterm birth, stillbirth, and the need for a neonatal intensive care unit. Conclusion: Severe COVID-19 infection in pregnancy is linked to pregnancy and birth complications, mainly among pregnant women with a positive medical history.",
"keywords": [
"COVID-19",
"pregnancy",
"pregnancy outcomes",
"risk factors",
"severity"
],
"content": "Introduction\n\nThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the source of the coronavirus disease 19 (COVID-19), a highly contagious and dangerous viral illness that led to a global pandemic a significant loss of human life.1 Severe acute respiratory syndrome coronavirus infection in pregnant women can cause either asymptomatic or symptomatic disease (mild, moderate, severe, and critical).2,3 Although the risk of developing severe illness is minimal, pregnant women with COVID-19 are more likely to do so than non-pregnant people. In addition, after adjusting for age, race/ethnicity, and underlying medical conditions, pregnant women had noticeably higher rates of intensive care unit (ICU) admission, mechanical ventilation, extracorporeal membrane oxygenation, and mortality.4\n\nContrary to pregnant and previously pregnant women without the illness, COVID-19-positive pregnant women had a higher risk of stillbirth and preterm birth.5 In addition, pregnancy-related physiological and immunological changes substantially impact the severity and effects of viral infections.6 For example, increased ACE2 expression, changes in the immune system to ensure the acceptance of the fetus, and changes in the respiratory system causing hyperventilation are all factors that could enhance vulnerability to COVID-19 infection during pregnancy.7\n\nCompared to non-pregnant women, pregnant women with COVID-19 are less likely to experience symptoms. Being overweight or obese, being older than 35 years, and having pre-existing comorbidities (hypertension, diabetes, lung diseases) are all variables that increase the risk of developing a severe illness.8 To determine how long COVID-19 patients stay in hospitals, it is critical to examine their risk factors. According to a study from China in 2022, patients aged ⥠45 and those with severe COVID-19 infection tended to stay longer in the hospital.9\n\nThe advantages of COVID-19 immunization, which include a decreased risk of severe illness and hospitalization for the pregnant woman and a reduced risk of hospitalization for the newborn in the first six months of life, should be discussed with expectant mothers.10 All eligible individuals, including those who are pregnant, nursing, or planning a pregnancy, should receive a COVID-19 vaccine or a series of vaccines, according to recommendations from the Society for Maternal-Fetal Medicine, American College of Obstetricians and Gynecologists, and CDC. If a person is qualified, the booster dosages are also included.11 The primary objectives of this study were to identify the most common symptoms associated with COVID-19 infection during pregnancy and assess the pre-existing risk of hospitalization, preterm delivery, and other complications related to COVID-19 illness, taking into account the severity of the disease.\n\n\nMethods\n\nThe institutional scientific and ethical committees of the Faculty of Pharmacy, Mustansiriyah University, and the Baghdad Health Directorate, Al-Karkh, formally approved the research protocol on November 10, 2021 (Ethics Board approval code: 2021099).\n\nSince the data has been anonymized, and because it is impracticable to obtain consent. The ethical committee waived the need for consent to participate. There is sufficient protection of participantsâ privacy and an adequate plan to protect the confidentiality of data.\n\nThis observational retrospective study was carried out in the obstetrics and gynecology wards of Al-Yarmouk Teaching Hospital and Karkh Hospital for childbirth between November 15, 2021, and August 27, 2022, where pregnant women with COVID-19 were admitted to the hospital for treatment or to give birth.\n\nThe sample size was determined and computed using the computer application G*Power 3.1.9.7 (RRID: SCR 013726). The smallest total sample size was 314 patients, with an effect size of 0.20 and 95% power at a two-tailed alpha of 0.05 and a 95% confidence interval (f). After reviewing the hospitalâs medical records or files, the biostatistics department provided 450 patient records; only 359 were selected for the research, while the rest fulfilled the exclusion criteria Figure 1.\n\n\n\n1. Pregnant women with COVID-19 positive PCR or Rapid test results with or without symptoms.\n\n2. Pregnant women were admitted for the treatment of COVID-19.\n\n3. Pregnant women with COVID-19 were admitted for delivery.\n\n\n\n1. Medical records of COVID-19-infected pregnant women with missing data (unknown PCR or rapid test)\n\n2. Pregnant women with other viral infections\n\n3. In vitro fertilization (IVF) pregnancy.\n\nInformation was gathered using the research teamâs specially created data collection form to match the studyâs objectives. Information on the patientâs age, gestational age, comorbidities (diabetes, hypertension, and anemia), and gynecological and obstetrical history (parity, gravidity, abortion, and the number of previous C-sections). Pregnancy outcomes and birth status (abortion, preterm birth, stillbirth, and requirement for NICU).\n\nThe severity of COVID-19 infection was categorized according to the following12â14:\n\nAsymptomatic: No symptoms.\n\nMild disease: Fever, cough, sore throat, nausea, vomiting, diarrhea, loss of taste or smell but no dyspnea; normal O2 saturation and standard chest X-ray.\n\nModerate disease: Symptoms of mild disease plus evidence of lower respiratory tract infection (exam and imaging), O2 saturation ⥠94% on room air.\n\nSevere disease: Symptoms of moderate disease but O2 saturation < 94%, PaO2/FiO2 < 300 mmHg, respiratory frequency > 30 breaths per minute, or lung infiltrates > 50%.\n\nCritical disease: Symptoms of severe disease but intubated with respiratory failure, septic shock, and multiorgan dysfunction.\n\nSince research operations, data collection, data entry, and data quality assurance are unplanned, retrospective cohort studies are frequently thought to have a higher bias. Using old data may affect any of these categories. But the authors of this study were keen to ensure that every eligible individual has an equal probability of selection. The research developed for settings considered, data completeness and quality assessment were conducted to ensure less bias during sample selection and study procedure.\n\nThe collected data were analyzed using SPSS version 25 statistical program for Windows (RRID: SCR 016479). The data are presented as mean ± standard deviation, number, and frequency. The chi-squared test was used to determine the significance of the associations between related categorical variables. Odds ratios were used to quantify the relationship between exposure and disease. Binary logistic regression was used to predict the relationship between independent and predicted variables. A two-sample t-test was used to determine the significance of differences between the means of the numerical data. A P-value less than 0.05 were considered a discrimination point for significance.\n\n\nResults\n\nThe mean age of COVID-19-infected pregnant women was 26.78-year ± 6.694 standard deviation (between 15-46 years), gestational age was 35.77-week ± 6.133 standard deviation (between 6-42 weeks), and birth weight at delivery was 2937.10 gram ± 607.457 standard deviation. Eight women were in the first trimester (2.2%), 15 in the second trimester (4.2%), and the majority of the cases (336) were in the third trimester (93.6%) as presented in Table 1.\n\nData present that 43.5% of the cases were asymptomatic, 35.4% were mild, 13.9% were moderate, and 7.2% were severe COVID-19 cases (Figure 2).\n\nStudy findings revealed that COVID-19-infected pregnant women with severe infection or aged â¥40 years had more extended hospital stays (â¥3 days) than those with non-severe illness or aged less than 40 years (P-value < 0.05) (Table 2).\n\n** P-value < 0.01 was considered highly significant.\n\nTable 3 shows those COVID-19-infected pregnant women who suffered from fever, cough, shortness of breath, and dyspnea had significantly more severe COVID-19 infection than those who did not suffer from the above clinical manifestations. P value = 0.001 for all conditions. Other symptoms didnât differ among severity P-value > 0.05.\n\n** P-value < 0.01 was considered highly significant.\n\nTable 4 showed that fever and shortness of breath were the most predictable signs and symptoms that may result in severe COVID-19 after adjustment for other symptoms that showed a significant association with the severity of COVID-19 condition according to the chi-square test presented in Table 3.\n\n* P-value < 0.05 was considered significant.\n\n** P-value < 0.01 was considered highly significant.\n\nDistribution of COVID-19 medications\n\nTable 5 shows that COVID-19-infected pregnant women who received multiple antibiotics, oxygen therapy, steroids, enoxaparin, and CPAP had significantly more severe COVID-19 infection than those who did not need the above treatments. P-value = 0.001 for all conditions.\n\n** P-value < 0.01 was considered highly significant.\n\nTable 6 presents the distribution of the recorded risk factors according to COVID-19 severity. Pregnant women with a history of diabetes, hypertension, and previous cesarean sections presented with a significant predominance of severe cases. The mean age of severe COVID-19-infected pregnant women was significantly higher than that of not severe cases (P-value = 0.001), while gestational age did not reveal any difference between severity types (P-value > 0.05).\n\n* P-value < 0.05 was considered significant.\n\n** P-value < 0.01 was considered highly significant.\n\nAfter adjustment for other risk factors, hypertension was found to be the most predictive risk factor for severe COVID-19 infection among pregnant women with a positive medical history (Table 7), according to the chi-square test presented in Table 6.\n\n** P-value < 0.01 was considered highly significant.\n\nFigure 3 shows that 5.1% of pregnancies ended with abortion, and 31.22% needed NICU and 6.4% ended with stillbirths among all the studied cases. Pregnancy outcomes according to the severity of covid-19 infection, as presented in Table 5, showed that 35% (7 out of 20) of stillbirth deliveries were in mothers with severe COVID-19 infection, which is significantly higher than stillbirth in non-severe cases, 65% (13 out of 294). (P-value = 0.001), (OR = 8.8), (95%CI = 3.1-24.8). Severe COVID-19-infected pregnant women delivered 13.9% (11 out of 79) of babies requiring NICU admission, significantly higher than mothers with non-severe infection (28%; 68 out of 239) (P-value = 0.014), (OR = 2.8), (95%CI = 1.2-6.4). Severe COVID-19-infected pregnant women delivered 19.4% (14 of 72) of preterm babies, significantly higher than mothers with non-severe infections (24.6%, 58/235) (P-value = 0.001), (OR = 5.16), (95%CI = 2.23-11.95) (see Table 8).\n\n* P-value < 0.05 was considered significant.\n\n** P-value < 0.01 was considered highly significant.\n\n\nDiscussion\n\nIn the current study, the mean age of the 359 pregnant women was approximately 26 years, and most of them were admitted to the hospital in the third trimester (gestational age of 35.77 weeks). These data agree with others, where the higher hospitalization rate of pregnant women with COVID-19 was in the third trimester.15,16\n\nRecently, human health has been severely threatened by COVID-19 infections that can range in severity from asymptomatic to severe (O2 saturation < 94%, hypoxia, lung involvement, and increased respiratory rate) or critical (multiorgan dysfunction and respiratory failure).17 According to recent research, pregnant women are more likely to experience severe illnesses, including the need for hospitalization, ICU admission, and mechanical ventilation.18 Asymptomatic and mild pregnancy cases (43.5%, 35.4%) in the current study represented the most hospitalized cases, while severe cases represented the least (7.2%) of the total cases. These results agree with previous studies, which revealed that most COVID-19-infected pregnant women were asymptomatic and had mild cases.19,20\n\nThe length of hospital stay (LOHS) was defined as the duration between the patientâs admission to the hospital and discharge. Data on duration of stay provide insights into the effectiveness of care over time, including associations between length of stay and hospital-acquired conditions (HACs). For example, extended hospital stays have been linked to high mortality rates for specific diseases and an increased risk of hospital-acquired infections. The current study showed that patients who experienced significantly extended LOHS (equal to or more than three days) were those with severe COVID-19 infection and those aged ⥠40 years. This result agrees with Wang et al. in 2022, who reported similar findings concerning the age and severity of illness with hospital stay.9\n\nThe most common symptoms of COVID-19 are fever, cough, dyspnea, and myalgia.5,21 COVID-19-infected pregnant women in this study had a fever, cough, shortness of breath, and other signs and symptoms such as nausea, vomiting, diarrhea, headache, weakness, and loss of smell or taste, which occurred in a small percentage of the included cases. These results agree with Gillian et al. in 2020, who reported that fever, cough, and SOB were the most common symptoms in pregnant women with COVID-19 infection.22 The common symptoms were more pronounced among severe COVID-19 infected pregnant women (P-value = 0.001) than those with mild or moderate cases. After adjusting for other signs and symptoms, fever and shortness of breath were the most predictable indicators of severity using binary logistic regression. Although Lian et al., in 2021, stated similar results regarding fever but argued by considering cough as a predictor of developing severe illness,23 the reason behind this argument may be due to pregnancy itself, as the shortness of breath may occur in normal pregnancy by excluding the pathological causes. It is a challenge for clinicians to differentiate.24\n\nThe data obtained in this study documented the use of multiple antibiotics in severe cases, representing 14.7% of total medications. Cephalosporins were the most commonly used group in 73% of the cases, followed by macrolides, doxycycline, and aminoglycosides. However, the use of carbapenems, quinolones, and penicillins was much lower. The use of multiple antibiotics among COVID-19-infected pregnant women, despite several guidelines that recommend antibiotics, except in the presence of secondary bacterial infection.25,26\n\nMedical oxygen is an essential medicine in treating COVID-19 and is used mainly to alleviate the severity of the infection.27 According to the Society for Maternal-Fetal Medicine, the target SpO2 for pregnant women should be higher than that recommended for the general population (SpO2 92%) owing to the increased O2 demand during pregnancy.28 Oxygen therapy was used in 23 patients in this study; of them, 22 patients had severe COVID-19 infection, all of whom showed improvement in clinical status and alleviation of the severity of the disease. According to numerous randomized trials, systemic corticosteroid therapy enhances clinical outcomes and lowers mortality in COVID-19 hospitalized patients who require supplementary oxygen.29â31 In contrast, systemic corticosteroids have not been proven beneficial and may even be harmful in COVID-19 hospitalized patients who do not need additional oxygen.32,33 In Pregnant women at risk of premature delivery, a brief course of betamethasone or Dexamethasone, which is known to cross the placenta, is frequently used to reduce neonatal complications of prematurity and decrease maternal mortality.34,35 In 22.8% of the cases included in this study, corticosteroids were used for both maternal and fetal purposes; however, pregnant women with COVID-19 infection who received corticosteroids had a more severe COVID-19 disease than those who did not (P = 0.001), so the more severe infection results in greater corticosteroid use, which is in line with national guidelines.\n\nInflammation and prothrombotic conditions have been linked to COVID-19, and fibrin breakdown products, fibrinogen, and D-dimer levels have increased.36 Some studies have linked the elevation of these markers to worse clinical outcomes.37,38 In addition, pregnant women have a higher risk of thromboembolism than non-pregnant women because pregnancy is a hypercoagulable state.39 Although there are no data for or against the use of anticoagulant therapy in the context of COVID-19 during pregnancy, the American College of Obstetricians and Gynecologists (ACOG) advises that VTE prophylaxis can be considered for pregnant women hospitalized with COVID-19, especially for those with severe disease. In the current study, enoxaparin was used in 31.5% of the cases, and its use was significantly associated with severe COVID-19 in pregnant women compared to those with non-severe disease (P = 0.001). Moreover, patients with COVID-19 successfully used CPAP to avoid endotracheal intubation. However, there is little information regarding the use of CPAP in expecting mothers with acute respiratory failure (ARF) caused by SARS-CoV-2 pneumonia.40 In this study, CPAP use was significantly associated with severe COVID-19 in pregnant women (P = 0.001), and there was no need for it in the non-severe cases.\n\nNumerous risk factors have been linked to the progression of COVID-19 into a severe and critical stage, including advanced age, male sex, and underlying comorbidities such as hypertension, diabetes, obesity, chronic lung diseases, heart, liver, and kidney diseases, tumors, and pregnancy.41,42 In the current study, COVID-19-infected pregnant women with a history of diabetes, hypertension, and previous cesarean section presented with a significant predominance of severe cases. In addition, the mean age of severe COVID-19-infected pregnant women was significantly higher (32.19 years) than that of non-severe patients. Allotey et al. 2020 mentioned that pregnant women with diabetes, hypertension, BMI > 30, and age > 35 years tend to have severe COVID-19 infection.5 Pregnant women with previous C-sections tend to have a severe infection, which may be due to the increased number of C-sections accompanied by additional complications.43 Also, individuals with COVID-19 with high blood pressure are related to higher mortality; the severity of COVID-19 may be exacerbated by inflammation and dysfunction of the immune system, gastrointestinal tract, and renin-angiotensin-aldosterone system; in addition, changes in innate cellular immunity contribute to high glucose environment, besides to more virulent growth of microbes.44,45\n\nAfter adjusting for risk factors that showed a significant association with the severity of infection (hypertension, diabetes, and previous C-section), predictions of risk factors were made using binary logistic regression to determine the most predictable risk factor. The test revealed that the incidence of COVID-19-infected pregnant women presenting with hypertension and diabetes developing severe infection was 9.173 and 4.297, respectively. These results are in agreement. In addition, Lian et al., in 2021, stated that patients with advanced age, H.T., and hypertension tend to have more severe COVID-19 infection.23\n\nBecause COVID-19 affects both the mother and the fetus and negatively impacts pregnancy outcomes, pregnant women with the infection need particular care and attention.46 This study explored the association between the severity of COVID-19 and adverse pregnancy outcomes. Among the entire study population, 6.3% (n = 20) of pregnant women had a stillbirth, 25% (n = 79) were admitted to the NICU, 4.8% (n = 16) had an abortion, and 22.6% (n = 72) had preterm delivery. There were 26 cases of COVID-19-infected pregnant women who presented with severe infection (delivery occurred in 25 patients) and had a higher rate of stillbirth deliveries (7 out of 25). In addition, admission to the NICU was higher among severe COVID-19-infected pregnant women (11 out of 24) and had preterm delivery (14 out of 25) than among non-severe cases. These results agree with those of Dileep et al. in 2022, who reported a strong association between the severity of COVID-19 infection and preterm birth, NICU admission, and low birth weight.46 Another study reported severe COVID-19 disease in pregnant women was associated with preterm and stillbirth deliveries.47\n\nThe collected data were retrospectively limited to two hospitals during one year. As a result, some of the collected data (particularly laboratory investigations) were not generally distributed in the patientâs records. In addition, the medical staff did not document important information, such as weight and body mass index (BMI), which strongly contributes to severity.\n\n\nConclusions\n\nMost COVID-19-infected pregnant women tend to have asymptomatic or mild cases of infection. However, severe COVID-19 illness is linked to pregnancy and birth complications, mainly among pregnant women with a positive medical history.",
"appendix": "Data availability\n\nZenodo: Underlying data for the impact of COVID-19 severity on pregnancy outcomes among Iraqi women: a retrospective observational study, https://doi.org/10.5281/zenodo.7607351. 48\n\nThis project contains the following underlying data:\n\nâ Article data.xlsx (underlying data of COVID-19 cases)\n\nZenodo: STROBE checklist for: The impact of COVID-19 severity on pregnancy outcomes among Iraqi women: a retrospective observational study, https://doi.org/10.5281/zenodo.7607382. 49\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe author would like to thank Mustansiriyah University in Baghdad, Iraq, for its support in the present work, and special thanks to all field participants.\n\n\nReferences\n\nShahreen MA, Khan S, Kazmi A, et al.: COVID-19 infection: Origin, transmission, and characteristic of human coronaviruses. J. Adv. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nPacker CH, Zhou CG, Hersh AR, et al.: Antenatal corticosteroids for pregnant women at high risk of preterm delivery with COVID-19 infection: a decision analysis. Am. J. Perinatol. 2020; 37(10): 1015â1021. PubMed Abstract | Publisher Full Text\n\nBoelig RC, Aagaard KM, Debbink MP, et al.: Society for Maternal-Fetal Medicine Special Statement: COVID-19 research in pregnancy: progress and potential. Am. J. Obstet. Gynecol. 2021; 225(6): B19âb31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHan H, Yang L, Liu R, et al.: Prominent changes in blood coagulation of patients with SARS-CoV-2 infection. Clin. Chem. Lab. Med. 2020; 58(7): 1116â1120. PubMed Abstract | Publisher Full Text\n\nGuan W-j, Ni Z-y, Hu Y, et al.: Clinical characteristics of coronavirus disease 2019 in China. N. Engl. J. Med. 2020; 382(18): 1708â1720. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTang N, Bai H, Chen X, et al.: Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. J. Thromb. Haemost. 2020; 18(5): 1094â1099. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLópez Y, Paloma MJ, Rifón J, et al.: Measurement of prethrombotic markers in the assessment of acquired hypercoagulable states. Thromb. Res. 1999; 93(2): 71â78. PubMed Abstract | Publisher Full Text\n\nFaverio P, Ornaghi S, Stainer A, et al.: Feasibility of CPAP application and variables related to worsening of respiratory failure in pregnant women with SARS-CoV-2 pneumonia: Experience of a tertiary care centre. PLoS One. 2021; 16(10): e0258754. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbbas SH, Abbas RS, Nafea LT: Severity and Risk of Death Due to COVID 19. Al Mustansiriyah Journal of Pharmaceutical Sciences. 2020; 20(4): 1â12. Publisher Full Text\n\nGao Y, Ding M, Dong X, et al.: Risk factors for severe and critically ill COVID-19 patients: a review. Allergy. 2021; 76(2): 428â455. PubMed Abstract | Publisher Full Text\n\nKaplanoglu M, Bulbul M, Kaplanoglu D, et al.: Effect of multiple repeat cesarean sections on maternal morbidity: data from southeast Turkey. Med. Sci. Monit. 2015; 21: 1447â1453. Publisher Full Text\n\nPeng M, He J, Xue Y, et al.: Role of Hypertension on the Severity of COVID-19: A Review. J. Cardiovasc. Pharmacol. 2021; 78(5): e648âe655. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRajana VK: Immune dysfunction in diabetes mellitus (DM). Int. J. Health Sci. Res. 2017; 7(12): 256â275.\n\nDileep A, ZainAlAbdin S, AbuRuz S: Investigating the association between severity of COVID-19 infection during pregnancy and neonatal outcomes. Sci. Rep. 2022; 12(1): 3024. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWei SQ, Bilodeau-Bertrand M, Liu S, et al.: The impact of COVID-19 on pregnancy outcomes: a systematic review and meta-analysis. CMAJ. 2021; 193(16): E540âE548. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalih OS: Underlying data for The impact of COVID-19 severity on pregnancy outcomes among Iraqi women: a retrospective observational study.2023. Publisher Full Text\n\nSalih OS: STROBE checklist for: The impact of COVID-19 severity on pregnancy outcomes among Iraqi women: a retrospective observational study.2023. Publisher Full Text"
}
|
[
{
"id": "193261",
"date": "15 Aug 2023",
"name": "Hayder Adnan Fawzi",
"expertise": [
"Reviewer Expertise Pharmaceutical science",
"clinical pharmacy",
"pharmacogenetics",
"medicine",
"and clinical trials."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe impact of COVID-19 severity on pregnancy outcomes among Iraqi women: a retrospective observational study\nMethods: Data collection\n\nWhere the information came from the patients? Datasheet or by other means? Please emphasize more about the methods of gathering information. Regarding âEthical considerationsâ\nThere are two institutes that approved the study, yet only one ethical approval is present, please provide both ethical approval codes, and submit the ethical approval as underlying data.\nRegarding the title of âTable 1â\nNeeds adjustment since it includes information regarding the neonate, it is better to useâ Table 1. Neonatal and maternal characteristicsâ\nRegarding table 3, 5, 6\nCalculate the percentage by column not by raw, since you omitted the number that indicates the no signs or symptoms (e.g., fever vs. no fever), for more clarity. In addition, some of the variables violate the principle that applied to the proper use of chi-square. How did you handle a sample with an expected frequency of less than 5%? Like Nausea, Diarrhea, and others?\nRegarding table 4\nReport using OR and its 95% confidence interval and remove the term Exp(β) and change it to OR.\nThis sentence âTable 4 showed that fever and shortness of breath were the most predictable signs and symptoms that may result in severe COVID-19 after adjustment for other symptoms that showed a significant association with the severity of COVID-19 condition according to the chi-square test presented in Table 3.â\nFirst, the chi-square test used is lower accuracy compared to logistic regression in Table 4, so please focus on using the outcome reported in Table 4. I would prefer to use only one statistical test (in Table 4) and remove the statistical analysis in Table 3, and merge the descriptive with the logistic analysis in one table.\nThe same note applied to tables 6 and 7 (merge both and report using logistic analysis)\nRegarding figure 3\nDraw using publication-level figures like GraphPad prism, and SAS.\nRegarding the result\nToo many tables that can be easily merged into fewer tables.\nYou did not use multivariate analysis which is lacking in your results and that affects your conclusion, if possible include them in your final analysis.\nDiscussion\nPlease start the discussion with the main subject of the article i.e. pregnancy outcomes and the impact of COVID-19 on it.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "206633",
"date": "08 Oct 2023",
"name": "Erin Oakley",
"expertise": [
"Reviewer Expertise Systematic review",
"meta-analysis",
"maternal health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst, I commend the authors on a thoughtful and thorough study of COVID-19 severity in pregnancy. The analysis is well done and will be useful to inform the body of knowledge around risk factors for severe COVID-19 (and related poor pregnancy outcomes).\nOne overall question I had when reading the paper is, do you have any insight as to what variant(s) of SARS-CoV-2 was circulating in Iraq at the time of the study? It seems that the data was collected in late 2021 through mid-2022, so this context may be relevant to note somewhere in the paper (perhaps in the discussion). There has been some evidence that the Delta variant in particular was associated with poor pregnancy outcomes.\n\nIn the abstract, I am unclear about what the conclusion statement \"mainly among pregnant women with a positive medical history\" means. Could you please clarify does this mean among pregnant women with pre-existing comorbidities?\nIn the introduction, it seems like these two statements (from paragraph 1 and paragraph 3, respectively) are in conflict: \"Although the risk of developing severe illness is minimal, pregnant women with COVID-19 are more likely to do so than non-pregnant people.\" (paragraph 1) \"Compared to non-pregnant women, pregnant women with COVID-19 are less likely to experience symptoms.\" (paragraph 3) Are these two statements based on conflicting evidence from different studies? Or, did you find in the literature that pregnant women were less likely to develop symptoms but more likely to develop severe symptoms rather than mild ones? It would be helpful to clarify.\nIn the section \"Characteristics of COVID-19 infected pregnant women\", it would be helpful to confirm:\nIs the gestational age given the age at infection onset (rather than pregnancy outcome)? Why only 283 observations for birthweight? Could you disaggregate Table 1 by disease severity? This would be an interesting addition.\n\nAlso, is it possible to share more about the distribution of participants by reason for admission? Based on your inclusion criteria, women could be recruited to the study based on admission for treatment of COVID-19 or admission for delivery (or both?).\nNoting a small typo in Figure 2 (should read \"severe\" in the yellow category).\nIn the section \"Length of hospital stay according to age and severity of COVID-19 infected pregnancy women\", it would be helpful to confirm:\nHow did you treat observations that were hospitalized for childbirth but were also infected (i.e., those asymptomatic cases or cases with more mild infection)? It looks like this table includes both admissions for childbirth and admissions for COVID-19 together. Understandably, it may be difficult to disentangle reasons for admission if someone has COVID-19 at the time of labor and delivery.\nIn the section \"Distribution of signs and symptoms of COVID-19 infected pregnant women according to the severity\": These are interesting findings. I wonder if your point would be better illustrated in Table 3 if you changed the way percentages are reported. Right now, the table shows that 87.2% of those with fever had a non-severe infection and 12.8% of those with fever had a severe infection. However, the point you make in the paragraph above Table 3 is that those who had severe infections were more likely to have a fever. I suggest instead of presenting the % column how it currently is, you consider instead presenting the % of participants in each severity category who had that symptom. For e.g.: 92% of participants with severe infection had fever vs. 50% of participants with non-severe infection. (Same suggestion for Table 5, Table 6, Table 8).\nWould it be possible to add confidence intervals to the last column of Table 4?\nIn the section \"Distribution of risk factors of COVID-19 [in] pregnant women according to the severity\": These findings are really interesting and well-presented. A few points to clarify:\nFor diabetes, does this include pre-existing diabetes prior to pregnancy, gestational diabetes, or both? For hypertension, does this include pre-existing hypertension prior to pregnancy, pregnancy-related hypertension, or both? How is anemia defined? You mention that mean maternal age is higher among severe cases than non-severe, but this is not included in Table 6 or Table 7. Did you consider including this in your risk factor analysis?\nIn the section \"Pregnancy outcomes and birth status among COVID-19 infected pregnant women\" the same recommendation as above, perhaps consider sharing the percentage of pregnancies that ended in stillbirth among severe and non-severe cases (per Table 8: 7/25 = 28% for severe and 13/333 = 4% for non-severe) rather than the percentage of total stillbirths that occurred in each group. I think this would help to illustrate your findings a bit better that adverse outcomes are more common among the severe cases than among the non-severe.\nThank you for sharing the data for this study -- this is well-presented and very informative. A few points to clarify when looking at the data compared to the study:\nIn the data, there are 20 stillbirths/359 observations (5.6%) and 16 abortions/359 observations (4.5%). This conflicts with Figure 3 (which shows 6.4% stillbirth and 5.1% abortion). Are some participants excluded from Figure 3 (e.g., perhaps due to loss of follow-up?) If so, this would be helpful to clarify in the paper or in the notes about how many pregnancies were followed through until the endpoint. Also, perhaps you could add an indicator to the dataset for \"loss to follow-up prior to delivery\" if this is the case so that others can reproduce the results. Further, the key at the top of the file lists 2 values for \"Preterm\" (1=yes, 2=no), but there are actually three values presented in this column (0, 1, 2). Also one further clarification for the data, what is the difference in the variable \"pregnancy outcome\" between 0 = \"no pregnancy complication\" and 2 = \"healthy\"? This would be useful for readers to interpret the data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "210968",
"date": "17 Oct 2023",
"name": "Joao Eudes Magalhaes",
"expertise": [
"Reviewer Expertise Neurology"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI received this manuscript on pregnancy outcomes and their correlation with the severity of COVID with enthusiasm. Although there is already a substantial body of literature published on the subject, it is always important to understand regional realities.\nHowever, the reading was challenging, primarily due to English grammatical errors. This will require special attention from the authors in a new version.\nThe Introduction needs to be rewritten to better express what has already been published on the topic. I especially suggest that the authors include the various existing systematic reviews on COVID in pregnant women. It is also important to clarify the objectives.\nIn the methods section, it was not clear what estimation was used for sample size calculation.\nIn the results section, some data are redundantly presented in both the text and the tables and graphs, so I suggest keeping only the text for greater clarity.\nNotwithstanding, most of the data analyses were not adequately presented for a more detailed assessment of this study. In several instances, the authors assumed a result contrary to what is demonstrated. For example, â(...) in Table 5, showed that 35% (7 out of 20) of stillbirth deliveries were in mothers with severe COVID-19 infection, which is significantly higher than stillbirth in non-severe cases, 65% (13 out of 294)â. How can 35% be significantly higher than 65%? This same premise was used to indicate several other comparisons throughout the results.\nUnfortunately, it was not possible to analyze the discussion and conclusion once the authors used wrong interpretations even if they were based on correctly presented results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-220
|
https://f1000research.com/articles/12-218/v1
|
27 Feb 23
|
{
"type": "Research Article",
"title": "The irritable bowel syndrome among adults in Qatif, Saudi Arabia: prevalence and impact on health-related quality of life, by gender and age",
"authors": [
"Ali Hassan A. Alnasser",
"Mohammed Sheker H. Al Kalif",
"Muneera Ahmed O. Alrowaila",
"Fatimah Mahdi A. Alhomaidi",
"Hasheem Murtada S. Alalsayedsalih",
"Ali Ahmad A. Alkabah",
"Ali Jaffar Q. Almanyan",
"Ali Hasan M. Al Zawwad",
"Hussain Tawfeeq A. Alalwan",
"Ahmed Tariq T. Bu Hassan",
"Komail Abdulaziz S. Al Abbad",
"Hassan Ayman H. Alibrahim",
"Ali Abdullah D. Awkar",
"Kauthar Jaffar A. Altawfiq",
"Fadel Ali M. Almosa",
"Mohammed Sheker H. Al Kalif",
"Muneera Ahmed O. Alrowaila",
"Fatimah Mahdi A. Alhomaidi",
"Hasheem Murtada S. Alalsayedsalih",
"Ali Ahmad A. Alkabah",
"Ali Jaffar Q. Almanyan",
"Ali Hasan M. Al Zawwad",
"Hussain Tawfeeq A. Alalwan",
"Ahmed Tariq T. Bu Hassan",
"Komail Abdulaziz S. Al Abbad",
"Hassan Ayman H. Alibrahim",
"Ali Abdullah D. Awkar",
"Kauthar Jaffar A. Altawfiq",
"Fadel Ali M. Almosa"
],
"abstract": "(1) Background: Even though irritable bowel syndrome (IBS) is not life-threatening, it can have a significant impact on a patient's daily activities and quality of life. Studies indicate that IBS is more prevalent in women than in men. The mechanism, etiology, and signs of IBS are not well understood, but there is evidence that gender and age is a biological characteristic that affects these subjects. This influence on the quality of life of Saudi adults by gender and age has not previously been compared. (2) Methods: A snowball sampling techniques of 279 people in Qatif, Saudi Arabia, is surveyed online to assess health-related quality of life (HRQoL). The Short SF-36 was self-administered to assess HRQoL; logistic regression models, Mann-Whitney, and the Kruskal-Wallis were used. SPSS version 23.0 was used to store and analyze all data. (3) Results: The prevalence of IBS among Saudi adults was 17.6%. IBS has a statistically significant association with age and gender which is more common among older women. IBS participants have poorer HRQoL than the overall population, particularly in Physical Component Score (PCS). (4) Conclusions: IBS is more prevalent among Saudi adults in Qatif and has a significant negative impact on the HRQoL, particularly among elderly women.",
"keywords": [
"IBS",
"IBS-QOL",
"SF-12",
"IBS-SSS",
"HRQoL",
"gastrointestinal disease",
"gastrointestinal disorder",
"GI disorders"
],
"content": "Introduction\n\nIrritable bowel syndrome (IBS) is the most frequently diagnosed digestive disorder.1 It is a symptom-based condition characterized by abdominal pain or discomfort and altered bowel habits in the absence of another disease causing these symptoms.1 IBS is one of the most prevalent disorders of gut-brain interactions (previously referred to as functional gastrointestinal disorders).2 Moreover, it is estimated to affect approximately one in ten people worldwide.2 Global prevalence estimates for IBS range widely from 5.7% to 34.0%, depending on the methodology utilized.3 IBS is more common in Westerners (between 10% and 15%) than in Asians (1% to 10%).4\n\nSubsequently, it affects 3.8% of people worldwide, and costs â¬8 billion in Europe and $10 billion in the US in direct and indirect healthcare costs.5 IBS is a prevalent disorder that has a significant impact on quality of life and accounts for a significant proportion of healthcare costs.6 IBS has a significant impact on the individual's health-related quality of life (HRQoL) and incurs significant costs, both in terms of healthcare delivery and in terms of the economy and society.2 Psychological comorbidities and extraintestinal somatic symptom reporting are common in IBS patients, lowering the quality of life, productivity, health impairment, and healthcare utilization.5 11% is the global prevalence of irritable bowel syndrome (IBS), according to a systematic review and meta-analysis, but this varies considerably depending on geographic region, diagnostic criteria used to define IBS, minimum symptom duration required, age, and gender.3 About half to two-thirds of people with IBS have significant psychological or social issues.7 Female and male IBS patients differ in symptomatology, comorbidity with other chronic pain syndromes and psychiatric disorders, and serotonergic medication efficacy, highlighting the need for gender-specific treatment.8 Psychological interventions are associated with an improvement in HRQoL in all domains.9 Populations in Arab countries have received comparatively less research attention. No research from any Arab country was included in a recent meta-analysis of the global prevalence of IBS.3 IBS prevalence data from Saudi Arabia and other Middle Eastern nations is limited. Nevertheless, to the best of our knowledge, no study has examined the effects of IBS on health-related quality of life in Saudis based on gender or age. The purpose of this research is to assess the effects of IBS on patients' quality of life based on age and gender among Saudi adults in Qatif, Saudi Arabia.\n\n\nMethods\n\nThe study was reviewed and approved by the Institutional Review Board Committee of Qatif Health Network, Qatif, Eastern Province, Saudi Arabia. The committee, on behalf of the Institutional Review Board, approved the research (Reference Number QCH-SREC0 36/2022). This study was conducted following the Declaration of Helsinki, electronic informed consent was obtained from each participant before starting the investigation. Participants could withdraw from the survey at any moment without providing any justification. The participants responded anonymously to the online survey by filling up an informed consent letter in the first section of the e-questionnaire. In the consent form, all the participants were provided with information concerning the research purpose, confidentiality of information, and the right to revoke their participation without prior justification.\n\nThis observational cross-sectional study was carried out the Qatif, Saudi Arabia, from November 15 to November 20, 2022, utilizing a Google Form self-administered survey circulated across a variety of social networks. Participants had to be Saudi citizens, at least 18 years old, willing to take part, and able to understand Arabic were the inclusion criteria. Exclusion criteria included being under 18, using psychotropic medications, and having a history of mental illness.\n\nSnowball sampling was used to gather Saudi population data. The e-questionnaire included an informed consent form, and participants consented to the survey after reading it. Participants were requested to share the e-questionnaire on social media. A web-based questionnaire was voluntary and non-commercial.\n\nThe first section of the questionnaire assessed respondents' general characteristics such as gender, age, educational level, job status, monthly income, and IBS status. The second section of the questionnaire focused on quality of life by employing the Short Form 12-Item Health Survey (SF-12), a self-report instrument to assess health-related quality of life (HRQoL) that includes 12 questions from which physical and mental components Summary can be derived. The theoretical range is 0 to 100, with higher scores indicating improved HRQoL.10\n\nHealth-related quality-of-life (HRQoL) measures like the SF-12 assess mental and physical functioning across two weeks. This scale has 12 items from the Short Form 36 health status questionnaire (SF-36). The SF-12 has two subscales: the Mental Component Summary (MCS) and the Physical Component Summary (PCS). Each subscale has a value between 0 and 100, with higher scores indicating better quality of life. Cronbach's α for MCS = 0.76; PCS =0.89) shows the SF-12's reliability and validity. Total scores indicating higher quality of life were measured by setting two criteria (poor and good), and 100 points were divided into an average form so that less than 50 score is poor HRQoL and 50 or more score is good HRQoL.10\n\nThe Statistical Package for Social Sciences (SPSS) version 23.0 IBM, Chicago, IL, USA, was used to store and analyze all data. To describe the demographic characteristics of the Saudi population, descriptive analyses were carried out. To investigate the probable causes of IBS, logistic regression models were used. Logistic regression models' odds ratios (OR) and 95% confidence intervals were examined. The gender and age variations in HRQoL were examined using the Mann-Whitney test and the Kruskal-Wallis test. P-values of equal or less than 0.05 were regarded as statistically significant (two-sided tests).\n\n\nResults\n\nA total of 279 respondents completed the online questionnaire (response rate:69.75%). The mean age in the sample of eligible respondents was 38.38 years old (SD = 12.62 years, range 18-76 years old). Most of the sample was female (N=157, 56.3%), young adults (N=143, 51.3%, range 18-39 years old), had bachelor's degrees (N=183, 65.6%), were employed (N=167, 59.9%), and had monthly income ($1500 - $5,000) (N=149, 53.4%). Most participants with IBS were female (N=32, 20.4%), middle-aged (N=23, 19.2%, range 40-59 years old), and had a bachelor's degree (N=39, 21.3%). (Table 1). IBS was found to be prevalent in 17.6% of participants, with a higher rate of prevalence among older adults 31.3%, and more in women 20.4% (Figure 1).\n\nOverall, the total HRQoL, MCS, and PCS were (42.8, 47.5, and 38.1, respectively). In contrast, overall HRQoL, MCS, and PCS scores for responders with IBS were (38.4, 41.4, and 35.4, respectively) (Figure 2).\n\nThe impact of irritable bowel syndrome on health-related quality of life were presented in Table 2. In the univariate logistic regression models, HRQoL (OR=0.332, 95% CI: 0.135-0.818) and similarly, PCS (OR=0.409, 95% CI: 0.215-0.778) were significantly associated with IBS in Saudi public.\n\n* P-Value â€â0.05.\n\n** P-Value < 0.01.\n\nTable 3 demonstrates the associations between gender and age and IBS. In the univariate logistic regression models, females (OR=2.067, 95% CI: 1.017-4.199) and similarly old-aged-adults (OR=4.235, 95% CI: 1.201-14.938) were significantly associated with IBS in the Saudi public.\n\n* P â€â0.05.\n\nIn Table 4, the differences in health-related quality of life by gender and age were shown. The female was statistically significantly different from the Saudi public in terms of physical composite summary scores (PCS). On the other hand, old adults in the Saudi public were statistically significantly different in their mental composite summary (MCS).\n\na Mann-Whitney test.\n\nb Kruskal Wallis Test.\n\n* P-Value â€â0.05.\n\n** P-Value < 0.01.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first study among Saudi adults in Qatif to use the SF-36, a dependable and valid instrument, to evaluate the impact of irritable bowel syndrome (IBS) on patients' quality of life depending on age and gender among Saudi adults. IBS impacts physical, emotional, and social functioning without causing mortality.11 HRQOL and age/gender in IBS patients are poorly studied. This paper seeks to evaluate the influence of IBS on the quality of life of Saudi adults in Qatif, Saudi Arabia.\n\nOur study found that the prevalence of IBS among Saudi adults is 17.6%, which is lower than that found in a 2019 Saudi national study,12 which is consistent with previous local studies,13â15 and approximately 10-20% of adults globally,16,17 and more than the global IBS prevalence of 3,8%.1 In our study, females were two times more likely to get IBS in the Saudi population (OR=2.067, 95% CI: 1.017-4.199). Consistent with previous research,3 the current study found that the women-to-men IBS prevalence ratio was 1.5:1. Several studies from the West and Saudi Arabia show that women suffer from IBS at a higher rate than men.18â20 In contrast, numerous Asian-based studies have found no gender differences in the prevalence of IBS.20 This result is inconsistent with the findings of a prior systematic review and meta-analysis that included 56 studies and 1,88,229 potential participants21 and is incompatible with the Saudi study.12 The explanations are given for sex differences in physiological factors such as gastrointestinal transit time, visceral sensitivity, central nervous system pain processing, and specific effects of sex hormones on gut function. Further factors such as sex differences in stress reactivity, neuroendocrine, and autonomic nervous system may play a role.18 In addition, investigations carried out in Asian nations like India22 and Taiwan23 have indicated a higher male or equal gender prevalence of IBS. These gender discrepancies might be partially explained by cultural influences as well as the technique of the study.\n\nAccording to our findings, we found a statistically significant association between age and IBS. People who were old-aged adults were four times significantly associated with IBS in the Saudi public (OR=4.235, 95% CI: 1.201-14.938). This result is inconsistent with the findings of a previous meta-analysis that did not find a statistically significant association between age and IBS3 and is inconsistent with the Saudi study.12 Most of the IBS prevalence in the present study were old adults participants, between the ages of 60 and 80, which is inconsistent with the reported literature,24,25 but consisted of systematic national health surveys data from six large studies including (4.7 million people) conducted in the United States.26\n\nThis study indicates conclusively that participants with IBS had a significantly poorer HRQoL than non-IBS based on age and gender. The participants with IBS in this study had poorer physical health functioning than the general population in Saudi Arabia. From these data, it is unclear whether psychological factors or physical causes contribute more to the gastrointestinal distress experienced by people with IBS. Recently, the IBS severity score was a strong negative correlation with HRQoL.27 Several studies have shown that people with IBS have considerably lower SF-36 scores than the entire US population, healthy Europeans, and healthy US college students.28â30 People who suffer from IBS have a lower HRQoL experience than the general population as a whole.9 As compared to healthy members of the Saudi population, those with IBS were found to have significantly lower HRQoL than those without the condition as measured with the short-scale SF-36 (P < 0.05). Hahn et al.31 discovered that the general health condition of IBS patients in the United Kingdom and the United States was significantly worse than that of the general populations in both countries.\n\nUsing a web-based survey methodology to collect data has several strengths: 1) The information was taken from a representative sample of the inhabitants of the city of Qatif in eastern Saudi Arabia. 2) the data was gathered quickly. This study had several limitations: 1) Participants first took part in an online survey study from one city only, so we cannot generalize to all Saudis. 2) Most problem with the web-based method is that you can't get too much clinical data. 3) In addition, there is a possibility of bias if certain individuals do not engage in an online panel owing to technological difficulties. 4) Due to the cross-sectional nature of this study, it is impossible to establish a cause-and-effect relationship. 5) In addition, the data for this study came from self-administered questionnaires, which are inherently subjective and, as a result, have the potential to result in some bias. 6) IBS is an episodic disorder with symptoms that may vary over time, sampling patients at a particular time point is a possible limitation of this and other study methodologies. We recommend conducting an expanded Saudi national study that includes all regions of Saudi Arabia, and we also recommend using the Rome III questionnaire for the diagnosis of IBS. Finally, future research is required to establish a causal relationship between IBS and mental and physical variables, comorbidities, and HRQoL.\n\n\nConclusions\n\nIrritable bowel syndrome (IBS) is more prevalent in elderly adults (17.3%), and it is associated with gender and age; women have twice the risk, and the elderly have four times the risk. Furthermore, this study demonstrates that IBS has a negative impact on Health-related quality of life (HRQoL), decreasing it by 30% overall and almost 41% in the Physical component score (PCS). The HRQoL of people who suffer from IBS can be enhanced by looking beyond age and gender.\n\n\nAuthor contributions\n\nConceptualization, A.H.A.A; methodology, A.H.A.A; software, A.H.A.A; validation, A.H.A.A; formal analysis, A.H.A.A; investigation, A.H.A.A, M.S.H.A., M.A.O.A., F.M.A.A., H.M.S.A., A.A.A.A., A.J.Q.A., K.J.A.A., A.H.M.A., H.T.A.A., A.T.T.B., K.A.S.A., H.A.H.A., and A.A.D.A.; resources, A.H.A.A; data curation, A.H.A.A; writingâoriginal draft preparation, A.H.A.A; writingâreview and editing, A.H.A.A; visualization, A.H.A.A; supervision, F.A.M.A., and A.H.A.A; project administration, A.H.A.A; All authors have read and agreed to the published version of the manuscript.",
"appendix": "Data availability\n\nZenodo. The irritable bowel syndrome among adults in Qatif, Saudi Arabia: prevalence and impact on health-related quality of life, by gender and age. DOI: https://doi.org/10.5281/zenodo.7599804. 32\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC BY 4.0 Public domain dedication).\n\n\nAcknowledgments\n\nWe would like to thank the Eitharqatif committee for helping to distribute the questionnaire.\n\n\nReferences\n\nOka P, et al.: Global prevalence of irritable bowel syndrome according to Rome III or IV criteria: a systematic review and meta-analysis. Lancet Gastroenterol. Hepatol. 2020; 5: 908â917. Publisher Full Text\n\nBlack CJ, Ford AC: Global burden of irritable bowel syndrome: trends, predictions and risk factors. Nat. Rev. Gastroenterol. Hepatol. 2020; 17: 473â486. Publisher Full Text\n\nLovell RM, Ford AC: Global Prevalence of and Risk Factors for Irritable Bowel Syndrome: A Meta-analysis. Clin. Gastroenterol. Hepatol. 2012; 10: 712â721.e4. Publisher Full Text\n\nChang F-Y, Lu C-L, Chen T-S: The current prevalence of irritable bowel syndrome in Asia. J. Neurogastroenterol. Motil. 2010; 16: 389â400. Publisher Full Text\n\nShiha MG, Aziz I: Review article: Physical and psychological comorbidities associated with irritable bowel syndrome. Aliment. Pharmacol. Ther. 2021; 54: S12âS23.\n\nHorwitz BJ, Fisher RS: The Irritable Bowel Syndrome. N. Engl. J. Med. 2001; 344: 1846â1850. Publisher Full Text\n\nZamani M, Alizadeh-Tabari S, Zamani V: Systematic review with meta-analysis: the prevalence of anxiety and depression in patients with irritable bowel syndrome. Aliment. Pharmacol. Ther. 2019; 50: 132â143. Publisher Full Text\n\nMulak A, Taché Y, Larauche M: Sex hormones in the modulation of irritable bowel syndrome. World J. Gastroenterol: WJG. 2014; 20: 2433â2448. Publisher Full Text\n\nCassar GE, Youssef GJ, Knowles S, et al.: Health-Related Quality of Life in Irritable Bowel Syndrome: A Systematic Review and Meta-analysis. Gastroenterol. Nurs. Off. J. Soc. Gastroenterol. Nurses Assoc. 2020; 43: E102âE122. Publisher Full Text\n\nAlnasser AHA, et al.: Impact of COVID-19 severity on health-related quality of life among Saudi adult patients. Infez. Med. 2022; 30: 223â230.\n\nGralnek IM, Hays RD, Kilbourne A, et al.: The impact of irritable bowel syndrome on health-related quality of life. Gastroenterology. 2000; 119: 654â660. Publisher Full Text\n\nAlqahtani NH, Mahfouz MEM: The Prevalence and Risk Factors of Irritable Bowel Syndrome in Saudi Arabia in 2019. Int. J. Prev. Med. 2022; 13: 13. Publisher Full Text\n\nAlaqeel MK, Alowaimer NA, Alonezan AF, et al.: Prevalence of irritable bowel syndrome and its association with anxiety among medical students at King Saud bin Abdulaziz University for Health Sciences in Riyadh. Pak. J. Med. Sci. 2017; 33: 33.\n\nIbrahim NKR, Battarjee WF, Almehmadi SA: Prevalence and predictors of irritable bowel syndrome among medical students and interns in King Abdulaziz University, Jeddah. Libyan J. Med. 2013; 8: 21287. Publisher Full Text\n\nArishi AM, et al.: Irritable bowel syndrome: prevalence and risk factors in Jazan Region, Saudi Arabia. Cureus. 2021; 13.\n\nLee S-Y, et al.: Irritable bowel syndrome is more common in women regardless of the menstrual phase: a Rome II-based survey. J. Korean Med. Sci. 2007; 22: 851â854. Publisher Full Text\n\nHeidelbaugh JJ, Stelwagon M, Miller SA, et al.: The spectrum of constipation-predominant irritable bowel syndrome and chronic idiopathic constipation: US survey assessing symptoms, care seeking, and disease burden. Am. J. Gastroenterol. 2015; 110: 580â587. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChang L, Heitkemper MM: Gender differences in irritable bowel syndrome. Gastroenterology. 2002; 123: 1686â1701. Publisher Full Text\n\nAlButaysh OF, AlQuraini AA, Almukhaitah AA, et al.: Epidemiology of irritable bowel syndrome and its associated factors in Saudi undergraduate students. Saudi J. Gastroenterol. Off. J. Saudi Gastroenterol. Assoc. 2020; 26: 89.\n\nKim YS, Kim N: Sex-Gender Differences in Irritable Bowel Syndrome. J. Neurogastroenterol. Motil. 2018; 24: 544â558. Publisher Full Text\n\nLovell RM, Ford AC: Effect of gender on prevalence of irritable bowel syndrome in the community: systematic review and meta-analysis. Off. J. Am. Coll. Gastroenterol. ACG. 2012; 107: 991â1000. Publisher Full Text\n\nJain AP, Gupta OP, Jajoo UN, et al.: Clinical profile of irritable bowel syndrome at a rural based teaching hospital in central India. J. Assoc. Physicians India. 1991; 39: 385â386.\n\nLu C-L, et al.: Current patterns of irritable bowel syndrome in Taiwan: the Rome II questionnaire on a Chinese population. Aliment. Pharmacol. Ther. 2003; 18: 1159â1169. Publisher Full Text\n\nGwee K-A, Lu C-L, Ghoshal UC: Epidemiology of irritable bowel syndrome in Asia: something old, something new, something borrowed. J. Gastroenterol. Hepatol. 2009; 24: 1601â1607. Publisher Full Text\n\nAmin HS, et al.: The prevalence of irritable bowel syndrome among Saudi population in Riyadh by use of Rome IV criteria and self-reported dietary restriction. Saudi J. Gastroenterol. Off. J. Saudi Gastroenterol. Assoc. 2021; 27: 383â390. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSandler RS: Epidemiology of irritable bowel syndrome in the United States. Gastroenterology. 1990; 99: 409â415. Publisher Full Text\n\nAndrae DA, Patrick DL, Drossman DA, et al.: Evaluation of the Irritable Bowel Syndrome Quality of Life (IBS-QOL) questionnaire in diarrheal-predominant irritable bowel syndrome patients. Health Qual. Life Outcomes. 2013; 11: 208â212. Publisher Full Text\n\nWhitehead WE, Burnett CK, Cook EW, et al.: Impact of irritable bowel syndrome on quality of life. Dig. Dis. Sci. 1996; 41: 2248â2253. Publisher Full Text\n\nChassany O, Marquis P, Fraitag B: European psychometric validation of a specific quality of life questionnaire in functional digestive disorders. Therapie. 1995; 50: A35.\n\nStrassels SA, Hahn BA: Short-form 36 (SF-36) scores in patients with irritable bowel syndrome (IBS) compared with US norms. Gastroenterology. 1600 JOHN F KENNEDY BOULEVARD, STE 1800: WB SAUNDERS CO-ELSEVIER INC; 1997; vol. 112. : A832âA832.\n\nHahn BA, Yan S, Strassels S: Impact of Irritable Bowel Syndrome on Quality of Life and Resource Use in the United States and United Kingdom. Digestion. 1999; 60: 77â81. Publisher Full Text\n\nAlnasser AHA: The irritable bowel syndrome among adults in Qatif, Saudi Arabia: prevalence and impact on health-related quality of life, by gender and age.2023. Publisher Full Text"
}
|
[
{
"id": "180713",
"date": "05 Jul 2023",
"name": "Andrew McCombie",
"expertise": [
"Reviewer Expertise Cross sectional studies",
"quality of life",
"irrititable bowel syndrome"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for the opportunity to peer review this manuscript. Please elaborate on the following:\nI do not understand Table 4. Is this comparing people in these subgroups with IBS to the general population? Or all people from the study to the general population? And who are the \"Saudi Public\"? Are these the people who are the control population within the survey for this study? Overall, Table 4 needs a bit more explanation.\n\nIn your conclusion you state \"Irritable bowel syndrome (IBS) is more prevalent in elderly adults (17.3%)\" yet I cannot see that figure of 17.3% anywhere in the results.\n\nYou also need to be careful about talking about \"risk\" in your conclusion. You have not calculated risk ratios but rather odds ratios. This is not a cohort study so you by definition have not calculated differences in risk.\n\nDid you consider calculating a multivariate model? If you chose not to perform one for some reason, it should be stated in the limitations. I suspect it is underpowered but you do need to make this clear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
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https://f1000research.com/articles/12-218
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https://f1000research.com/articles/11-1522/v1
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15 Dec 22
|
{
"type": "Software Tool Article",
"title": "SnakeMAGs: a simple, efficient, flexible and scalable workflow to reconstruct prokaryotic genomes from metagenomes",
"authors": [
"Nachida Tadrent",
"Franck Dedeine",
"Vincent Hervé",
"Nachida Tadrent",
"Franck Dedeine"
],
"abstract": "Background: Over the last decade, we have observed in microbial ecology a transition from gene-centric to genome-centric analyses. Indeed, the advent of metagenomics combined with binning methods, single-cell genome sequencing as well as high-throughput cultivation methods have contributed to the continuing and exponential increase of available prokaryotic genomes, which in turn has favored the exploration of microbial metabolisms. In the case of metagenomics, data processing, from raw reads to genome reconstruction, involves various steps and software which can represent a major technical obstacle. Methods: To overcome this challenge, we developed SnakeMAGs, a simple workflow that can process Illumina data, from raw reads to metagenome-assembled genomes (MAGs) classification and relative abundance estimate. It integrates state-of-the-art bioinformatic tools to sequentially perform: quality control of the reads (illumina-utils, Trimmomatic), host sequence removal (optional step, using Bowtie2), assembly (MEGAHIT), binning (MetaBAT2), quality filtering of the bins (CheckM), classification of the MAGs (GTDB-Tk) and estimate of their relative abundance (CoverM). Developed with the popular Snakemake workflow management system, it can be deployed on various architectures, from single to multicore and from workstation to computer clusters and grids. It is also flexible since users can easily change parameters and/or add new rules. Results: Using termite gut metagenomic datasets, we showed that SnakeMAGs is slower but allowed the recovery of more MAGs encompassing more diverse phyla compared to another similar workflow named ATLAS. Conclusions: Overall, it should make the reconstruction of MAGs more accessible to microbiologists. SnakeMAGs as well as test files and an extended tutorial are available at https://github.com/Nachida08/SnakeMAGs.",
"keywords": [
"Snakemake",
"metagenomics",
"microbiology",
"genomics",
"bioinformatics",
"microbial ecology"
],
"content": "Introduction\n\nOver the last years, microbial ecology has progressively made the transition from gene-centric to genome-centric analyses,1 allowing the clear assignment of (sometimes novel) microbial taxa to specific functions and metabolisms.2â5 Indeed, technical and technological progresses such as binning methods applied to metagenomics,6 single-cell genome sequencing7 as well as high-throughput cultivation methods8 have contributed to the continuing and exponential increase of available prokaryotic genomes.9 This is particularly true for metagenomics that offers the possibility to reconstruct metagenome-assembled genomes (MAGs) on a large scale and from various environments, and thus has generated a huge amount of new prokaryotic genomes.10,11\n\nAlthough the use of MAGs in microbial ecology is becoming a common practice nowadays, processing raw metagenomic reads up to genome reconstruction involves various steps and software which can represent a major technical obstacle, especially for non-specialists. To face this problem, several workflows such as MetaWRAP,12 its Snakemake version called SnakeWRAP,13 ATLAS14 and more recently MAGNETO,15 have been developed to automatically reconstruct genomes from metagenomes. However, these workflows contain various modules and perform more tasks than only generating MAGs. For instance, they will taxonomically assign the metagenomic reads, create gene catalog or perform functional annotations. They rely on numerous dependencies, require significant computational resources and regenerate a lot of outputs which are not essential to most research projects. To simplify this procedure and make it more accessible while remaining efficient, reproducible and biologically relevant, we developed with the popular Snakemake workflow management system,16 a configurable and easy-to-use workflow called SnakeMAGs to reconstruct MAGs in just a few steps. It integrates state-of-the-art bioinformatic tools to sequentially perform from Illumina raw reads: quality filtering of the reads, adapter trimming, an optional step of host sequence removal, assembly of the reads, binning of the contigs, quality assessment of the bins, taxonomic classification of the MAGs and estimation of the relative abundance of these MAGs.\n\n\nMethods\n\nOur tool was built by integrating a set of software needed to process metagenomic datasets, utilizing Snakemake. There are no additional equations/maths needed to recreate this tool.\n\nThe workflow has been developed with the workflow management system Snakemake v7.0.016 based on the Python language. Snakemake enables reproducible and scalable data analyses as well as an independent management of the required software within a workflow. SnakeMAGs is composed of two main files:\n\nThe Snakefile, named âSnakeMAGs.smkâ, contains the workflow script. It is divided into successive rules which correspond to individual steps. Our workflow includes a total of 15 distinct rules. Each rule requires input files and relies on a single software installed independently when starting the workflow in a dedicated conda v4.12.0 environment. At the end of each rule, output files will be generated in a dedicated folder, as well as a log file (stored in the logs folder) summarizing the events of the software run and a benchmark file (stored in the benchmarks folder) containing the central processing unit (CPU) run time, the wall clock time and the maximum memory usage required to complete the rule. Thanks to Snakemake wildcards, our rules are generalized, so one can process multiple datasets in parallel without having to adjust the source code manually.\n\nThe configuration file,40 named âconfig.yamlâ, is used to define some variable names (e.g. names of the input files), paths (e.g. working directory, location of the reference databases), software parameters and computational resource allocations (threads, memory) for each of the main steps.\n\nTo run the workflow, the user only requires Snakemake. It can be easily installed, for instance via Conda, as explained in the GitHub repository:\n\n\n\nAfter that, the user will only have to edit the config file (an example is provided on the GitHub repository) and then run SnakeMAGs:\n\n\n\nDuring the first use of the workflow, a dedicated Conda environment will be installed for each of the bioinformatic tool to avoid conflict. Then the input files will be processed sequentially. Output files will be stored in eight dedicated folder: logs, benchmarks, QC_fq (containing FASTQ files), Assembly, Binning, Bins_quality (all three containing FASTA files), Classification (containing FASTA files and text files with the taxonomic information), and MAGs_abundances (text files).\n\nThe workflow has been successfully used on a workstation with Ubuntu 22.04 as well as on high-performance computer clusters with Slurm v18.08.7 and SGE v8.1.9.\n\nThe minimal system requirements to run the workflow will depend on the size of the metagenomic dataset. Small datasets (e.g. the test files provided on the GitHub repository) have been successfully analyzed on a workstation with an Intel Xeon Silver 4210, 2.20GHz (10 cores/20 threads) processor and 96GB of RAM. Larger datasets should be processed on cluster computing or within a high-performance infrastructure. For instance, performance evaluation of publicly available metagenomes (see below) was performed on a computer cluster under CentOS Linux release 7.4.1708 distribution with Slurm 18.08.7, on a node possessing an Intel Xeon CPU E7-8890 v4, 2.20GHz (96 cores/192 threads) and 512 GB RAM.\n\nSnakeMAGs integrates a series of bioinformatic tools to sequentially perform from Illumina raw reads: quality filtering of the reads with illumina-utils v2.12,17 adapter trimming with Trimmomatic v0.3918 (RRID:SCR_011848), an optional step of host sequence removal (e.g. animal or plant sequences) with Bowtie2 v2.4.519 (RRID:SCR_016368), assembly of the reads with MEGAHIT v1.2.920 (RRID:SCR_018551), binning of the contigs with MetaBAT2 v2.1521 (RRID:SCR_019134), quality assessment of the bins with CheckM v1.1.322 (RRID:SCR_016646), classification of the MAGs with GTDB-Tk v2.1.023 (RRID:SCR_019136) and estimation of the relative abundance of these MAGs with CoverM v0.6.1. An overview of the workflow is presented in Figure 1.\n\nThe names of the software used for each step are showed in parentheses.\n\n\nUse cases\n\nTo demonstrate the benefits and potential of our workflow, we compared it to another Snakemake workflow named ATLAS v2.9.1.14 To produce a fair comparison, ATLAS was run with the MEGAHIT assembler, without co-binning and dereplicating only 100% similar MAGs. To test these two workflows, we downloaded and analyzed ten publicly available termite gut metagenomes (accession numbers: SRR10402454; SRR14739927; SRR8296321; SRR8296327; SRR8296329; SRR8296337; SRR8296343; DRR097505; SRR7466794; SRR7466795) from five studies24â28 and belonging to ten different termite species.\n\nSnakeMAGs requires only a limited number of inputs files: the raw metagenomic reads in FASTQ format from the 10 above-mentioned metagenomes, a FASTA file containing the adapter sequences,40 a YAML configuration file specifying the variable names, paths and computational resource allocations (available on the GitHub repository and on Zenodo), and here since we worked with host-associated metagenomes a FASTA file containing the termite genome sequences.39 Regarding the outputs, SnakeMAGs produced quality-controlled FASTQ files without adapters nor termite sequences, in the QC_fq folder. Then the reads assembled into contigs and scaffolds (FASTA files) were saved in the Assembly folder. Products of the binning procedure were stored in the Binning folder. Bins with >50% completeness and <10% contamination (according to CheckM) were considered as MAGs and stored in the Bins_quality folder. Subsequently, the results of the MAGs classification and relative abundance estimation were sent to the Classification and MAGs_abundances folders, respectively. ATLAS requires similar input files and produces, among others, similar outputs files.\n\nATLAS appeared to be faster than SnakeMAGs to reconstruct MAGs from metagenomes (Figure 2A). However, SnakeMAGs always recovered more MAGs (>50% completeness and <10% contamination according to CheckM) per metagenome or at least as much as ATLAS (Figure 2B). From the ten metagenomes, SnakeMAGs produced a total of 65 MAGs while ATLAS generated only 37 MAGs. Additionally, SnakeMAGs was able to recover MAGs encompassing a higher diversity of bacterial phyla (n = 15 phyla) compared to ATLAS (n = 11 phyla). Only one phylum, namely Patescibacteria, represented by a single MAG was recovered by ATLAS and not by SnakeMAGs. On the contrary, ATLAS failed to reconstruct MAGs belonging to Verrucomicrobiota, Planctomycetota, Synergistota, Elusimicrobiota and Acidobacteriota when SnakeMAGs succeeded (Figure 2C).\n\nA. CPU time (in seconds) required to process each metagenome. B. Number of MAGs reconstructed from each metagenome. On both boxplots, gray lines link the result obtained with ATLAS and the one obtained with SnakeMAGs for each of the 10 analyzed termite metagenomes. C. Number of bacterial MAGs at the phylum level recovered from each workflow.\n\n\nDiscussion\n\nUsing metagenomic datasets from the gut of various termite species, our analyses revealed that while being slower, SnakeMAGs allowed the recovery of more MAGs encompassing more diverse phyla compared to ATLAS, another similar Snakemake workflow. More importantly our results showed that SnakeMAGs was able to recover MAGs encompassing the major bacterial phyla found in termite guts,29,30 and that some of these phyla were not recovered by ATLAS. Indeed, taxa belonging to Verrucomicrobiota,31 Planctomycetota,30,32 Synergistota,33 Elusimicrobiota34 and Acidobacteriota35,36 have been repeatedly found in the gut of various termite species. As such, they would represent relevant targets for genome-centric analyses of the termite gut microbiota. Therefore, we showed that SnakeMAGs has the potential to retrieve quantitatively more genomic information from metagenomes but also to extract genomic features of biological interest.\n\nThanks to the inherent flexibility of Snakemake, SnakeMAGs offers the possibility to the users to easily tune the parameters of the workflow (e.g. resource allocations for each rule, options of a specific tools) to adapt their analysis to the datasets and to the computational infrastructure. Additionally, advanced users will have the opportunity to edit or add new rules to the workflow. Regarding the future of SnakeMAGs, several avenues will be considered for the next versions of the workflow. Firstly, the workflow could give more freedom to the users by offering the choice of different tools to perform the same task (e.g. different trimming, assembly or binning software). Secondly, with the current emergence of metagenomic datasets generated with long-read DNA sequencing,37 it might be relevant to adjust our workflow for long-read sequencing technology by including specific bioinformatic tools for this technology.38 Meanwhile, since the majority of the metagenomic datasets have been and are still currently generated with Illumina short-read technology, SnakeMAGs can be widely used to explore the genomic content of various ecosystems via metagenomics.\n\n\nSoftware availability\n\nSource code available from: https://github.com/Nachida08/SnakeMAGs\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.7334838.39\n\nLicense: CeCILL v2.1",
"appendix": "Data availability\n\nTermite genome references used for removing host sequences and their Bowtie2 index are available at: https://zenodo.org/record/6908287#.Y1JLANJBzUR\n\nThe termite gut metagenomes analyzed in the present study are available on NCBI with the following accession numbers: SRR10402454; SRR14739927; SRR8296321; SRR8296327; SRR8296329; SRR8296337; SRR8296343; DRR097505; SRR7466794; SRR7466795.\n\nZenodo. Reconstruction of prokaryotic genomes from ten termite gut metagenomes using two distinct workflows: SnakeMAGs and ATLAS: https://doi.org/10.5281/zenodo.7334397. 40\n\n- SnakeMAGs_config.yaml (The configuration file used to analyze the 10 termite gut metagenomes with SnakeMAGs)\n\n- ATLAS_config.yaml (The configuration file used to analyze the 10 termite gut metagenomes with ATLAS)\n\n- MAGs_SnakeMAGs.zip (A zipped folder containing the genomes of the 65 MAGs reconstructed with SnakeMAGs)\n\n- MAGs_ATLAS.zip (A zipped folder containing the genomes of the 37 MAGs reconstructed with ATLAS)\n\n- taxonomic_assignment_MAGs.csv (A text file containing the taxonomic assignment of all the MAGs reconstructed by both workflows)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors thank Emmanuelle Morin and HélÚne Gardon for their valuable advice and feedback during the workflow development.\n\n\nReferences\n\nProsser JI: Dispersing misconceptions and identifying opportunities for the use of âomicsâ in soil microbial ecology. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nPasolli E, Asnicar F, Manara S, et al.: Extensive unexplored human microbiome diversity revealed by over 150,000 genomes from metagenomes spanning age, geography, and lifestyle. Cell. 2019 Jan; 176(3): 649â662.e20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUritskiy GV, DiRuggiero J, Taylor J: MetaWRAPâa flexible pipeline for genome-resolved metagenomic data analysis. Microbiome. 2018 Dec 15; 6(1): 158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrapohl J, Pickett B: SnakeWRAP: a Snakemake workflow to facilitate automated processing of metagenomic data through the metaWRAP pipeline [version 2; peer review: 1 approved]. F1000Res. 2022; 11(265). Publisher Full Text\n\nKieser S, Brown J, Zdobnov EM, et al.: ATLAS: a Snakemake workflow for assembly, annotation, and genomic binning of metagenome sequence data. BMC Bioinformatics. 2020 Dec 22; 21(1): 257. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChurcheward B, Millet M, Bihouée A, et al.: MAGNETO: An automated workflow for genome-resolved metagenomics. mSystems. 2022; 7(4): e00432âe00422.PubMed Abstract | Publisher Full Text | Free Full Text\n\nMölder F, Jablonski KP, Letcher B, et al.: Sustainable data analysis with Snakemake. F1000Res. 2021; Vol. 10:PubMed Abstract | Publisher Full Text | Free Full Text Reference Source\n\nEren AM, Vineis JH, Morrison HG, et al.: A filtering method to generate high quality short reads using Illumina paired-end technology. PLoS One. 2013; 8(6). Publisher Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014 Aug 1; 30(15): 2114â2120. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLangmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012 Apr 4; 9(4): 357â359. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi D, Liu CM, Luo R, et al.: MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 2015 May 15; 31(10): 1674â1676. PubMed Abstract | Publisher Full Text\n\nKang DD, Li F, Kirton E, et al.: MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies. PeerJ. 2019 Jul 26; 7: e7359. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParks DH, Imelfort M, Skennerton CT, et al.: CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015 Jul; 25(7): 1043â1055. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChaumeil PA, Mussig AJ, Hugenholtz P, et al.: GTDB-Tk v2: memory friendly classification with the Genome Taxonomy Database. Bioinformatics. 2022 Oct 11; btac672.Publisher Full Text\n\nCalusinska M, Marynowska M, Bertucci M, et al.: Integrative omics analysis of the termite gut system adaptation to Miscanthus diet identifies lignocellulose degradation enzymes. Communications Biology. 2020 Dec 1; 3(1): 275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoreira EA, Persinoti GF, Menezes LR, et al.: Complementary contribution of fungi and bacteria to lignocellulose digestion in the food stored by a neotropical higher termite. Front. Ecol. Evol. 2021 Apr 26; 9: 248. Publisher Full Text\n\nRomero Victorica M, Soria MA, Batista-GarcÃa RA, et al.: Neotropical termite microbiomes as sources of novel plant cell wall degrading enzymes. Sci. Rep. 2020 Dec 2; 10(1): 3864. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTokuda G, Mikaelyan A, Fukui C, et al.: Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher termites. Proc. Natl. Acad. Sci. 2018 Dec 18; 115(51): E11996âE12004.Publisher Full Text\n\nWaidele L, Korb J, Voolstra CR, et al.: Ecological specificity of the metagenome in a set of lower termite species supports contribution of the microbiome to adaptation of the host. Animal Microbiome. 2019 Dec 24; 1(1): 13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArora J, Kinjo Y, Å obotnÃk J, et al.: The functional evolution of termite gut microbiota. Microbiome. 2022 Dec; 10(1): 78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHervé V, Liu P, Dietrich C, et al.: Phylogenomic analysis of 589 metagenome-assembled genomes encompassing all major prokaryotic lineages from the gut of higher termites. PeerJ. 2020 Feb; 8: e8614. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWertz JT, Kim E, Breznak JA, et al.: Genomic and physiological characterization of the Verrucomicrobia isolate Diplosphaera colitermitum gen. nov., sp. nov., reveals microaerophily and nitrogen fixation genes. Appl. Environ. Microbiol. 2012 Mar 1; 78(5): 1544â1555. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKöhler T, Stingl U, Meuser K, et al.: Novel lineages of Planctomycetes densely colonize the alkaline gut of soil-feeding termites (Cubitermes spp.). Environ. Microbiol. 2008 May; 10(5): 1260â1270. PubMed Abstract | Publisher Full Text\n\nAhmad F, Yang G, Zhu Y, et al.: Tripartite symbiotic digestion of lignocellulose in the digestive system of a fungus-growing termite. Microbiology Spectrum. 2022 Oct 17; e01234âe01222. Publisher Full Text\n\nHerlemann DPR, Geissinger O, Ikeda-Ohtsubo W, et al.: Genomic analysis of âElusimicrobium minutum,â the first cultivated representative of the phylum âElusimicrobiaâ (formerly termite group 1). Appl. Environ. Microbiol. 2009 May 1; 75(9): 2841â2849. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHongoh Y, Deevong P, Inoue T, et al.: Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl. Environ. Microbiol. 2005 Nov 1; 71(11): 6590â6599. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBourguignon T, Lo N, Dietrich C, et al.: Rampant host switching shaped the termite gut microbiome. Curr. Biol. 2018 Feb; 28(4): 649â654.e2. PubMed Abstract | Publisher Full Text\n\nBickhart DM, Kolmogorov M, Tseng E, et al.: Generating lineage-resolved, complete metagenome-assembled genomes from complex microbial communities. Nat. Biotechnol. 2022 Jan 3; 40: 711â719. PubMed Abstract | Publisher Full Text\n\nFeng X, Cheng H, Portik D, et al.: Metagenome assembly of high-fidelity long reads with hifiasm-meta. Nat. Methods. 2022 Jun; 19(6): 671â674. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTadrent N, Dedeine F, Hervé V: SnakeMAGs (v1.0.2). [Code] Zenodo. 2022. Publisher Full Text\n\nTadrent N, Dedeine F, Hervé V:Reconstruction of prokaryotic genomes from ten termite gut metagenomes using two distinct workflows: SnakeMAGs and ATLAS. [Data]. Zenodo. 2022. Publisher Full Text"
}
|
[
{
"id": "158646",
"date": "16 Jan 2023",
"name": "Célio Dias Santos Júnior",
"expertise": [
"Reviewer Expertise Bioinformatics",
"microbiology",
"biochemistry",
"biotechnology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA method to bin MAGs from reads is described in the publication \"SnakeMAGs: a simple, efficient, flexible and scalable approach to rebuild bacterial genomes from metagenomes\" by Tradent et al. The use of this pipeline to standardize the work with MAGs and make it accessible to batch runs has an obvious benefit. Although the pipeline itself appears a little out of date in terms of conceptual work in the area, I think it has the potential to develop into a useful tool for bioinformatics in general with a few modifications. The critique I offer in this review is more directed at the pipeline's ongoing structural evolution than it is towards the actual article. Although the study reads extremely well, further work must be done to adequately support the use of SnakeMAGs.\n# Abstract\nThe authors here should have put greater emphasis on what may be anticipated in terms of the quality of the genomes and species retrieved. It is not favorable if you can recover more genomes but they are of inferior quality, smaller size, or skewed towards a certain species.\n# Methods\nI want to congratulate the authors for the open scientific component. The databases, code, and software were all presented in an organized manner. The documentation reads quite well and appears to be a crucial component of your work. Both the tool's installation and usage are quite well-explained and simple.\nAs an adept of collaborative research, I saw that your GitHub lacked some unit tests that would have allowed other contributors to repair bugs that the authors had little interaction with or had previously overlooked without jeopardizing the distribution. That is not a question in this assessment, but it may represent a future enhancement.\nI understand the authors' desire to keep the pipeline short to avoid several dependencies. However, it doesn't appear that the main pipeline is more effective or addresses that issue. For instance, ATLAS is faster, although relying on more dependencies. What benefit do SnakeMAGs have in this regard?\nDespite being condensed, SnakeMAGs does appear to skip certain necessary stages to produce better MAGs, including:\nUsing software that controls for chimera, such as metaMIC1, to fix contigs obtained after assembly;\n\nIncluding multiple binning systems which may seem like a delay but ensures the best bin for each species and even offers a better resolution at the strain level. For instance, using a next-generation binner, such as semiBin or VAMB, then afterward clustering the MAGs on an ANI basis to assure a higher resolution of the species found. Several of these techniques even permit the use of long reads, which may ultimately be advantageous;\n\nSince we now know that some of the species binned occasionally display under or overestimated completeness due to bias in the calculations using USCG, run a quality check of the bins using a more comprehensive approach than CheckM, for example, implementing software that addresses contamination, such as GUNC, or even more advanced updates of previous systems that now incorporate machine learning, such as CheckM2 , which does not present bias towards a taxonomy. It would be the best solution for this quality-checking step.\nIs there a plan to integrate these actions in the upcoming versions? How may these omitted stages affect the outcomes in the current SnakeMAGs version?\n# Results\nWhat was the reason for choosing termite gut metagenomes? Is there any indication of better sequencing, deeper sequencing, higher microbial diversity, or any other reason that led the authors to this choice?\nThe quality criteria used by authors to classify good genomes (>50% completeness and <10% contamination according to CheckM) is quite loose and not widely adopted. Parks et al. (2017)2 - one of the first works using MAGs and also where checkM was presented - used an estimated quality â¥50 (defined as completeness â 5 à contamination). In the present work, if a genome is in the bottom line, it would have an estimated quality of 0, which is far from the threshold Parks adopted. Other criteria are also quite important, e.g. the number of contigs, N50, and ambiguous base pairs. I recommend authors reassess these results using a more strict quality parameter and then report if their results are better or comparable to the other pipelines. I believe that if other binners were combined and similar bins merged as in Parks et al. (2017)2, SnakeMAGs would represent a game changer.\nThe fact that \"SnakeMAGs was able to recover MAGs encompassing a higher diversity of bacterial phyla\" is impressive, and I wonder what the average quality of these MAGs recovered in those phyla that ATLAS was not able to bin. Is there any chance that ATLAS discarded bad-quality MAGs that SnakeMAGs is assuming as correct?\nThe analysis regarding the memory needed by both systems is missing. The authors mentioned that the peak of memory usage is registered in the SnakeMAGs' logs, and a comparison with ATLAS needs seems important. The longer time for processing, if accompanied by a reduction in memory needs, still seems advantageous in my POV.\n# Discussion\nThe fact that the phyla recovered by SnakeMAGs match well with reports of microbial diversity in the samples analyzed are quite appealing to me. However, do the abundances of these species also vary largely? How abundant the phyla recovered only by SnakeMAGs are? Authors should explore this factor to explain if SnakeMAGs' advantage lies in binning genomes from rare species.\nIt makes me happy to know that the authors plan versions of the pipeline along with improvements. This spirit of continuous development usually ends up in great bioinformatics tools.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "9374",
"date": "27 Feb 2023",
"name": "Vincent Hervé",
"role": "Author Response",
"response": "# Abstract The authors here should have put greater emphasis on what may be anticipated in terms of the quality of the genomes and species retrieved. It is not favorable if you can recover more genomes but they are of inferior quality, smaller size, or skewed towards a certain species. Reply â We agree, it is indeed an important point. In the revised version of the manuscript, we show that the quality and genome size of the MAGs recovered by SnakeMAGs only did not differ from those belonging to phyla recovered by both SnakeMAGs and ATLAS. This is now specified in the abstract of the manuscript. # Methods I want to congratulate the authors for the open scientific component. The databases, code, and software were all presented in an organized manner. The documentation reads quite well and appears to be a crucial component of your work. Both the tool's installation and usage are quite well-explained and simple. Reply â We thank Reviewer 1 for these positive comments. As an adept of collaborative research, I saw that your GitHub lacked some unit tests that would have allowed other contributors to repair bugs that the authors had little interaction with or had previously overlooked without jeopardizing the distribution. That is not a question in this assessment, but it may represent a future enhancement. Reply â We thank Reviewer 1 for this suggestion. We will fully consider it for future enhancement. Meanwhile, users have the opportunity to open Issues on the GitHub page to report any bug or request. Additionally, the test files provided on GitHub allow the user to test our workflow before running real and large datasets. I understand the authors' desire to keep the pipeline short to avoid several dependencies. However, it doesn't appear that the main pipeline is more effective or addresses that issue. For instance, ATLAS is faster, although relying on more dependencies. What benefit do SnakeMAGs have in this regard? Reply â Our main goal was to design a simple and minimalist workflow, so a non-specialist can easily bin MAGs without having to choose among (or test and compare) the myriads of software currently available to perform the different steps of our workflow. We believe that the strength of SnakeMAGs in this regard is its simplicity. Using SnakeMAGs, a non-specialist user just needs to follow general instructions, while using ATLAS, such a user need to make decisive choices of various tools at many steps of the workflow, an approach that can easily become quite confusing. Despite being condensed, SnakeMAGs does appear to skip certain necessary stages to produce better MAGs, including: Using software that controls for chimera, such as metaMIC1, to fix contigs obtained after assembly;  Including multiple binning systems which may seem like a delay but ensures the best bin for each species and even offers a better resolution at the strain level. For instance, using a next-generation binner, such as semiBin or VAMB, then afterward clustering the MAGs on an ANI basis to assure a higher resolution of the species found. Several of these techniques even permit the use of long reads, which may ultimately be advantageous;  Since we now know that some of the species binned occasionally display under or overestimated completeness due to bias in the calculations using USCG, run a quality check of the bins using a more comprehensive approach than CheckM, for example, implementing software that addresses contamination, such as GUNC, or even more advanced updates of previous systems that now incorporate machine learning, such as CheckM2 , which does not present bias towards a taxonomy. It would be the best solution for this quality-checking step. Is there a plan to integrate these actions in the upcoming versions? How may these omitted stages affect the outcomes in the current SnakeMAGs version? Reply â We thank Reviewer 1 for these constructive comments. We acknowledge that the processing of metagenomic reads as well as binning are currently very active fields of research and thus, new tools have been published since we started working on our workflow. As mentioned in our Discussion, in the upcoming versions we plan to include alternative software to perform certain tasks and the binning step will be one of them with the addition of SemiBin. CheckM2 is currently in a preprint stage but we also plan to include it as soon as it will be peer-reviewed. Regarding the chimera, we felt that it was a significant gap in our workflow so we decided to release a version 1.1 of SnakeMAGs that now includes GUNC for genome quality evaluation. We also evaluated the impact of this tool on the MAGs generated by both ATLAS and SnakeMAGs. For the SnakeMAGs genomes, we found that 59 out of 65 MAGs, encompassing 13 phyla, passed the GUNC filtering step. With ATLAS, we found that 29 out of 37 MAGs, encompassing 9 phyla, passed the GUNC filtering step. In summary, a few MAGs generated by both workflows did not pass the GUNC quality criteria, but including GUNC does not change the major outcome of our comparison with ATLAS: SnakeMAGs produces more MAGs and more diverse MAGs than ATLAS. This analysis has now been included in the revised version of the manuscript. # Results What was the reason for choosing termite gut metagenomes? Is there any indication of better sequencing, deeper sequencing, higher microbial diversity, or any other reason that led the authors to this choice? Reply â It was indeed a deliberate choice to select termite gut metagenomes. We have been working on termite gut microbiota for many years so we are familiar with the microbial diversity present in such systems. Therefore, we have the expertise to properly evaluate the outputs of the workflows from a biological perspective. We would have been less confident with samples from other ecosystems (e.g. deep-sea sediments). Additionally, we would like to mention that termites are well-known to harbor a higher microbial diversity compared to other arthropods. We also selected samples encompassing hosts from different termite families and with different diets, two factors known to impact the gut microbial diversity. Therefore, we believe that termite gut metagenomes constitute relevant datasets to test our workflow. The quality criteria used by authors to classify good genomes (>50% completeness and <10% contamination according to CheckM) is quite loose and not widely adopted. Parks et al. (2017)2 - one of the first works using MAGs and also where checkM was presented - used an estimated quality â¥50 (defined as completeness â 5 à contamination). In the present work, if a genome is in the bottom line, it would have an estimated quality of 0, which is far from the threshold Parks adopted. Other criteria are also quite important, e.g. the number of contigs, N50, and ambiguous base pairs. I recommend authors reassess these results using a more strict quality parameter and then report if their results are better or comparable to the other pipelines. I believe that if other binners were combined and similar bins merged as in Parks et al. (2017)2, SnakeMAGs would represent a game changer. Reply â Regarding the quality criteria, we used the criteria of âMedium quality MAGâ as defined by the Genomic Standards Consortium for the Minimum Information about a Metagenome-Assembled Genome (see Bowers et al, 2017, Nature Biotechnology). This is now specified in the revised version of the manuscript. However, we acknowledge that other authors have used higher quality standard. Following the reviewerâs suggestion, we reassessed our results using the estimated quality â¥50 (defined as completeness â 5 à contamination). As expected, using this higher quality threshold the number of recovered MAGs decreases compared to our initial criteria for both workflows: from 37 to 31 MAGs for ATLAS and from 65 to 46 MAGs for SnakeMAGs. Therefore, SnakeMAGs still allows the recovery of more MAGs than ATLAS. In terms of diversity, SnakeMAGs also recover more phyla (n = 13) than ATLAS (n = 10). In summary, the advantages of our workflow are robust to the MAG quality criteria. These results are now included in the revised version of the manuscript. It is noteworthy that in the version 1.1 of SnakeMAGs we have now implemented in the Quality assessment step an option to filter the MAGs according to this estimated quality criteria (completeness â 5 à contamination). This will allow users to freely select more stringent quality criteria if they want to. The fact that \"SnakeMAGs was able to recover MAGs encompassing a higher diversity of bacterial phyla\" is impressive, and I wonder what the average quality of these MAGs recovered in those phyla that ATLAS was not able to bin. Is there any chance that ATLAS discarded bad-quality MAGs that SnakeMAGs is assuming as correct? Reply â This is indeed an important point that is now reported in the revised version of the manuscript. Overall, we found no difference in MAG quality or genome size between the two workflows (Wilcoxon test, P = 0.15 for completeness; P = 0.60 for contamination and P = 0.64 for genome size). Regarding the MAGs generated by SnakeMAGs, we found no difference in MAG quality or genome size between the phyla also recovered by ATLAS and the phyla only recovered by SnakeMAGs (Wilcoxon test, P = 0.19 for completeness; P = 0.43 for contamination and P = 0.19 for genome size). Therefore, we found no evidence supporting the fact that ATLAS discarded bad-quality MAGs that SnakeMAGs is assuming as correct. These results are now included in the revised version of the manuscript. The analysis regarding the memory needed by both systems is missing. The authors mentioned that the peak of memory usage is registered in the SnakeMAGs' logs, and a comparison with ATLAS needs seems important. The longer time for processing, if accompanied by a reduction in memory needs, still seems advantageous in my POV. Reply â Following Reviewer 1 suggestion, we performed this comparison but found not significant difference in memory usage between the two workflows (Wilcoxon test, P = 0.393). This result is now specified in the revised version of the manuscript. # Discussion The fact that the phyla recovered by SnakeMAGs match well with reports of microbial diversity in the samples analyzed are quite appealing to me. However, do the abundances of these species also vary largely? How abundant the phyla recovered only by SnakeMAGs are? Authors should explore this factor to explain if SnakeMAGs' advantage lies in binning genomes from rare species. Reply â We thank Reviewer 1 for this relevant comment. We now further discuss this point in the revised version of the manuscript. We found no significant difference in the relative abundance (Wilcoxon test, P = 0.51) of these MAGs compared to the other phyla. However, it should be noted that although Verrucomicrobiota, Planctomycetota, Synergistota, Elusimicrobiota and Acidobacteriota have been reported among the major taxa in termite gut, they are usually less abundant than Spirochaetota, Firmicutes and Bacteroidota (Arora et al, 2022, Microbiome) that have been recovered by both workflows. This result strongly suggests that SnakeMAGs is indeed not restricted to the most abundant taxa."
}
]
},
{
"id": "159904",
"date": "15 Feb 2023",
"name": "Aram Mikaelyan",
"expertise": [
"Reviewer Expertise Entomology",
"Microbial Ecology",
"Microbial systematics",
"Symbiosis",
"Bioinformatics",
"Evolution"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe SnakeMAGs as a dedicated pipeline for the compositional binning of contigs generated from Illumina sequencing reads obtained from microbial communities. It integrates the various steps in the process of comparative analyses in metagenomes, starting with the assembly of reads generated from community DNA, to the compositional binning of the assembled contigs into high-quality metagenome assembled contigs (MAGs). I believe this pipeline represents a valuable addition to the community. I found the manuscript was easy to read and have the following comments for the authors:\nThe main advantage of Snake is that it is highly task-specific to the generation of high-quality MAGs from complex communities. It therefore can save considerable time and resources by avoiding steps in processing data (e.g. extensive functional or taxonomic annotations) that are irrelevant to the researcherâs objective.\nI would like the authors to expand on how their pipeline controls for chimeric sequences, since these may lead to an artefactual inflation of MAGs.\n\nAs a termite researcher, I am aware of the many quirks that termite gut microbiomes present, and the limitations of many data processing pipelines and databases when it comes to addressing those quirks. I was glad that the authors used this challenging community for their benchmarks, and impressed at the contrast between the results from SnakeMAGs and ATLAS. I will emphasize further that the apparent inability of ATLAS to report phyla such as Planctomycetota and Elusimicrobia, or reconstruct fewer MAGs from Fibrobacterota or Desulfobacterota, is not trivial. These bacterial members play major roles in the microbial ecology of the termite gut, and the SnakeMAGsâ ability to detect them highlights its superiority.\nI would like the authors to list/explain some factors could potentially account for this difference.\n\nI found the installation and usage on a Linux Mint server (with comparable processing power to the one used by the authors) to be straightforward. The dependencies are minimalistic and easy to install.\nI congratulate the authors on sharing this tool with the community and hope they find my comments useful.\nAram Mikaelyan\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "9375",
"date": "27 Feb 2023",
"name": "Vincent Hervé",
"role": "Author Response",
"response": "The authors describe SnakeMAGs as a dedicated pipeline for the compositional binning of contigs generated from Illumina sequencing reads obtained from microbial communities. It integrates the various steps in the process of comparative analyses in metagenomes, starting with the assembly of reads generated from community DNA, to the compositional binning of the assembled contigs into high-quality metagenome assembled contigs (MAGs). I believe this pipeline represents a valuable addition to the community. I found the manuscript was easy to read and have the following comments for the authors: 1. The main advantage of Snake is that it is highly task-specific to the generation of high-quality MAGs from complex communities. It therefore can save considerable time and resources by avoiding steps in processing data (e.g. extensive functional or taxonomic annotations) that are irrelevant to the researcherâs objective. I would like the authors to expand on how their pipeline controls for chimeric sequences, since these may lead to an artefactual inflation of MAGs. Reply â Indeed, this aspect was not considered in the first version of our workflow. Therefore, we decided to release a version 1.1 of SnakeMAGs that now includes GUNC, a software for detection of chimerism and contamination in prokaryotic genomes. We also evaluated the impact of this tool on the MAGs generated by both ATLAS and SnakeMAGs. For the SnakeMAGs genomes, we found that 59 out of 65 MAGs, encompassing 13 phyla, passed the GUNC filtering step. With ATLAS, we found that 29 out of 37 MAGs, encompassing 9 phyla, passed the GUNC filtering step. In summary, a few MAGs generated by both workflows did not pass the GUNC quality criteria. Importantly, this new analysis shows that including GUNC does not change the major outcome of our comparison with ATLAS: SnakeMAGs produces more MAGs and more diverse MAGs than ATLAS. This analysis has now been included in the revised version of the manuscript.  2. As a termite researcher, I am aware of the many quirks that termite gut microbiomes present, and the limitations of many data processing pipelines and databases when it comes to addressing those quirks. I was glad that the authors used this challenging community for their benchmarks, and impressed at the contrast between the results from SnakeMAGs and ATLAS. I will emphasize further that the apparent inability of ATLAS to report phyla such as Planctomycetota and Elusimicrobia, or reconstruct fewer MAGs from Fibrobacterota or Desulfobacterota, is not trivial. These bacterial members play major roles in the microbial ecology of the termite gut, and the SnakeMAGsâ ability to detect them highlights its superiority. I would like the authors to list/explain some factors could potentially account for this difference. Reply â A potential explanation for this difference can be the relative abundance of the phyla recovered only with SnakeMAGs compared to the relative abundance of the phyla recovered by both workflows. We now further discuss this point in the revised version of the manuscript. We found no significant difference in the relative abundance (Wilcoxon test, P = 0.51) of the MAGs belonging to phyla recovered only with SnakeMAGs compared to the phyla recovered by both workflows. However, it should be noted that although Verrucomicrobiota, Planctomycetota, Synergistota, Elusimicrobiota and Acidobacteriota have been reported among the major taxa in termite gut, they are usually less abundant than Spirochaetota, Firmicutes and Bacteroidota (Arora et al, 2022, Microbiome) that have been recovered by both workflows. This result suggests that SnakeMAGs is not restricted to the most abundant taxa unlike ATLAS, which could be relatively more prompted to recover only the most abundant microbial taxa. 3. I found the installation and usage on a Linux Mint server (with comparable processing power to the one used by the authors) to be straightforward. The dependencies are minimalistic and easy to install. I congratulate the authors on sharing this tool with the community and hope they find my comments useful. Reply â We thank Reviewer 2 for this positive feedback."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1522
|
https://f1000research.com/articles/10-1211/v1
|
26 Nov 21
|
{
"type": "Research Article",
"title": "The perceived impact of the COVID-19 pandemic on medical studentsâ future careers",
"authors": [
"Heba Mahjoub",
"Chirag Vasavda",
"Amanda Bertram",
"Ashwini Davison",
"Stephen Sozio",
"Chirag Vasavda",
"Amanda Bertram",
"Ashwini Davison",
"Stephen Sozio"
],
"abstract": "Background: The COVID-19 pandemic disrupted medical education on multiple levels, and medical students have been forced to adjust to distance learning, altered clinical opportunities, and standardized testing inconsistencies. We sought to identify the effects of these dramatic deviations on medical studentsâ career plans. Methods: We conducted a cross-sectional online survey of medical students between July 13, 2020, and September 9, 2020 in order to assess the implications of the COVID-19 pandemic on studentsâ career decisions. Descriptive statistics were calculated for all variables. Results: Of the 585 eligible medical students, we had a final sample of 76 responses (n=76) (13% response rate). Students felt neutral regarding having more time to explore research projects (Mean ± SD; 3.06 ± 1.18) and hobbies (3.43 ± 1.28). Most survey respondents somewhat disagreed that they considered quitting medical school during the pandemic (1.55 ± 1.10). Students somewhat agreed that they view the field of medicine more positively since the onset of the COVID-19 pandemic (3.60 ± 1.09). Respondents somewhat agreed that they would be unable to explore other specialties and find their best fit (3.55 ± 1.32). We found that the minority (4/66, 6%) of students had considered changing their specialty. Students felt neutral in terms of their Step 1 (3.25 ± 1.05) or Step 2 (2.81 ± 1.02) score deterring them from future career opportunities. Conclusions:  Most medical students have experienced barriers in their career pathway as a direct cause of COVID-19 restrictions on medical education, including the ability to explore different specialties to discover their best fit or find a chance to network with mentors. However, despite these obstacles, most students remain committed to medicine.",
"keywords": [
"medical education",
"COVID-19 pandemic",
"medical students",
"career planning"
],
"content": "Introduction\n\nThe Coronavirus disease-2019 (COVID-19) pandemic created unforeseen obstacles for humankind across the globe. Tasks that were once mundane have now become difficult or unsafe. Importantly, medical students have been uniquely affected. For many months, the Association of American Medical Colleges (AAMC) advised medical schools across the United States to pause in-person education in order to slow the spread of the virus.1 Most medical schools transitioned pre-clinical curricula to an online format, embracing learning from a distance. Unfortunately, clinical years, where medical students serve directly on medical teams rather than in a classroom, were more difficult to transfer online.2 To further complicate the situation, mandatory standardized tests such as USMLE Step 1 and Step 2 were postponed or cancelled for many students.3 Many medical students have also been unable to continue their scientific research and volunteer opportunities in the community. Such experiences are crucial in career development as they help shape studentsâ interests.\n\nOne learning theory that forms the theoretical framework for this study is connectivism, which emphasizes the importance of technology and socialization for building an educational framework.4 It especially highlights that learning is a continuous process that requires nurturing connections between study areas.5 Interestingly, this theory supports both virtual learning and in-person learning, muddying the potential outcomes of a transition from clinical rotations to e-learning. While different students have been affected by COVID-19 in various ways,6 the aggregate impact of the pandemic will resonate throughout both medicine and society for years to come. Hence, we sought to study the implications of the COVID-19 pandemic on students' career decisions. In particular, we aimed to investigate the impact of COVID-19 on medical studentsâ perspectives towards medicine. We explored whether the pandemic may have altered studentsâ professional and personal goals, such as whether interests in specialties had changed, whether they were questioning medicine as a career, and whether they felt well-educated on pandemics and infectious diseases. We also aimed to determine studentsâ reactions to changes in licensing exams and how this uncertainty might influence their careers.\n\n\nMethods\n\nThis research was acknowledged and exempted by the Johns Hopkins Institutional Review Board (IRB00248820). Prior to survey administration, participants received emails stating that their voluntary participation in the survey represented their informed written consent to participate.\n\nWe conducted a cross-sectional survey between July 13, 2020, and September 9, 2020. Requests for participation were sent out to Johns Hopkins students via Qualtrics® version XM using individual email links, followed by three reminder emails every 2-3 weeks. Medical students currently enrolled in Johns Hopkins University School of Medicine pursuing a Doctor of Medicine (MD), MD/Doctor of Philosophy (PhD), MD/ Master of Public Health (MPH), MD/Master of Business Administration (MBA) degree upon graduation were included. The survey items were developed to cover topics ranging from physical and emotional well-being during the pandemic, as well as perceptions of the effects on career development (Underlying data).7 Emotional well-being was ascertained according to validated assessment.8 Respondents rated statements according to a 5-point Likert scale, with a score of 1 representing âstrongly disagreeâ, 2 âsomewhat disagreeâ, 3 âneutralâ, 4 âsomewhat agreeâ, and 5 âstrongly agreeâ. Descriptive statistics were calculated for all variables using Qualtrics® version XM. version XM.\n\nAll individuals surveyed were medical students who were affected by changes to their medical education due to the COVID-19 pandemic. The study is sensitive to selection bias for students that responded to and completed the survey, potentially limiting the generalizability of our findings from all students. Additionally, our survey only reached medical students at a single institution and thus may not reflect the experience of students at other institutions. The study size was determined as the total number of medical students enrolled and eligible to receive the survey by email. All data were included in the analyses.\n\n\nResults\n\nOf the 585 eligible medical students, we received 83 total responses. Seven responses were excluded because only the first question was answered, leaving a final sample of 76 responses (n = 76) (13% response rate). Most respondents were women (43/76, 57%), and the average age was 25.68 (± 2.45 standard deviation (SD)). In this sample, 51% (39/76, 51%) were white, followed by 29% (22/76, 29%) Asian or Pacific Islander, 8% (6/76, 8%) multi-racial, and 8% (6/76, 8%) Black or African American. Many students had received prior degrees including two with PhDs (2/76, 2%), three with Master of Public Health (MPH)s (3/76, 4%), three with Master of Science (MS)s (3/76, 4%), and one with a Master of Arts (MA) (1/76, 1%). No students (0/71, 0%) were offered an early graduation option by their medical school at the height of the pandemic (Table 1).\n\nA series of COVID-19 related questions were included in the survey (Table 2) (Underlying data).7 When asked the question âAre there currently restrictions in the place where you live?â, 50% (50/100, 50%) of students responded âMy town/city is in the process of easing restrictionsâ, as well as 46 students (46/100, 46%) who responded âSocial isolation is recommended by state or local authorities. Of those who responded, 25% (25/71, 35%) had friends or family members who had been diagnosed with COVID-19, although none (0/71, 0%) were themselves diagnosed with COVID-19. In the overall cohort, most respondents (37/71, 52%) were leaving their home four or more times per week for work, shopping, or other reasons.\n\nSeveral survey questions (Table 3) covered student well-being. On average, students somewhat agreed with currently feeling that they have a definite role among family and friends (Mean ± SD; 3.89 ± 1.00), that they are useful to family and friends (3.80 ± 1.13), that they are being listened to (3.87 ± 0.92), and that they know what is going on with their family and friends (4.18 ± 0.86). Students felt neutral regarding having more time to explore research projects (3.06 ± 1.18) and hobbies (3.43 ± 1.28), as well as more time to spend with friends or family members (3.34 ± 1.37). Table 3 shows additional responses regarding student well-being. In a section of our survey that permitted free text, students shared their difficult experiences. An example of one experience was (Extended data)9:\n\nâI was recently diagnosed with major clinical depression. While being quarantined and having to contend with the ever more present issue of police brutality against BIPOC individuals [Black, Indigenous and People of Color] and systemic racism, I often feel exhausted, unmotivated, useless, and general sadness which makes it extremely difficult to wake up and go about my days.â\n\nStudents were also asked about their medical education prior to COVID-19 and their perceived obligations (Table 3). When questioned about the adequacy of their medical education regarding epidemics, they felt neutral (3.49 ± 1.08). Students felt neutral regarding their confidence in the healthcare system (3.34 ± 1.37) and their medical schoolâs administration (3.12 ± 1.33). Most survey respondents somewhat disagreed that they considered quitting medical school during the pandemic (1.55 ± 1.10). The students somewhat agreed that they view the field of medicine more positively since the onset of the COVID-19 pandemic (3.60 ± 1.09) and that it is their responsibility to volunteer during the pandemic (3.37 ± 1.12). Table 3 indicates additional responses covering medical education prior to COVID-19 and perceived obligations.\n\nThere were multiple survey questions regarding obstacles to mentorship and residency preparation (Table 3). Respondents somewhat agreed that they would be unable to explore other specialties and find their best fit (3.55 ± 1.32), as well as being unable to network with potential mentors (3.79 ± 1.15). Students were neutral regarding the level of comfort forming relationships with faculty members (3.11 ± 1.28), asking for letters of recommendation (2.97 ± 1.22), and level of competitiveness for their desired field (2.71 ± 1.08). In terms of their career, students were neutral that losing the ability to participate in away rotations would limit their career trajectory (2.89 ± 1.22), that they would not perform as well on graded clerkships as before (3.13 ± 1.37), and that they would not have the opportunity to complete sub-internships or advanced electives at their institution (3.18 ± 1.34).\n\nWe were also curious as to whether the pandemic had caused students to switch their preferred specialty and the effects of the pandemic on the standardized exams, Step 1 and Step 2. We found that the minority (4/66, 6%) of students had considered changing their specialty. For those who had considered changing, these swaps were from emergency medicine to neurosurgery, otolaryngology to internal medicine, neurosurgery to internal medicine, and internal medicine to anesthesiology (Figure 1). Six students (6/66, 9%) absolutely believed that the COVID-19 pandemic would negatively impact their current or future Step 1 score, while 13 (13/66, 19.6%) did not believe that it would. The majority (37/66, 56.0%) of students did not believe the pandemic had negatively affected their current or future Step 2 score. Neither group felt strongly that their Step 1 (3.25 ± 1.05) or Step 2 (2.81 ± 1.02) score would deter them from future career opportunities (Table 4). One student shared their experience with the testing sites during the pandemic (Extended data)9:\n\nâI think the biggest frustration for a lot of people has been uncertainty of their test date, as testing appointments continued to get cancelled last minute. It's incredibly frustrating and exhausting to prepare for months for a specific day, just to find out in the days before that you'll have to change your date and continue studying.â\n\n\nDiscussion/conclusion\n\nIn this cross-sectional survey, we explored the important topic of how the COVID-19 pandemic has influenced medical studentsâ professional goals. We analyzed 76 responses from diverse medical students with various educational backgrounds. Most students perceive barriers in their career pathway, including the ability to explore different specialties to discover their best fit or find a chance to network with mentors. However, despite these obstacles, most students feel strongly to continue in medicine on the same pathway that they began.\n\nWe found that many students lived in a town or city that was in the process of easing restrictions (50/100, 50%), but that social isolation was still recommended by state or local authorities (46/100, 46%). Some students (11/71, 16%) were even living with high-risk populations. These factors led medical schools to pause in-person learning. Though transitioning classes to an online format might seem effective, the impact of isolated learning has already taken an effect on students. Even when learning has continued in a digital, socially distanced format, studies have shown that students are experiencing disheartening decreases in overall work performance and emotional detachment.10 This is concerning, considering the highest quality of patient care comes from doctors working effectively as teams.11\n\nInterestingly, students in our study did not feel they had more or less time to spend on research projects (3.06 ± 1.18), on hobbies (3.43 ± 1.28), or with friends and family (3.34 ± 1.37). Although students did not feel an increase or decrease in time spent on research projects, they did feel strongly that other opportunities for career preparation were stunted. Respondents somewhat agreed that they would have difficulty exploring other specialties to find their best fit (3.55 ± 1.32), as well as being unable to network with potential mentors (3.79 ± 1.15). Overall, however, respondents were neutral when questioned about their competitiveness for their desired field (2.71 ± 1.08). Further, students might have felt that their Step 1 or Step 2 scores were affected by the pandemic, but students did not feel strongly that these scores would deter them from future career opportunities. These findings indicate that while certain experiences have been limited by the pandemic, students generally see themselves on the same path that they initially envisioned. We found that only the minority (4/66, 6%) of students had considered changing their specialty. This is less than a previous studyâs finding that one-fifth of medical students would change their choice of specialty.12 This study, by Byrnes et al., involved a larger sample size including students at various institutions, but the findings showed that region of the US did not alter whether the pandemic affected studentsâ specialty choices. Furthermore, our study found that students felt neutral (2.97 ± 1.22) in asking for letters of recommendation, whereas the respondents in the Byrnes et al. study posed that as their main cause of specialty change.\n\nIn addition, most survey respondents somewhat disagreed that they considered quitting medical school during the pandemic (1.55 ± 1.10). Coupled with the data that these students view the field of medicine more positively since the onset of the COVID-19 pandemic (3.60 ± 1.09), this suggests a resilient attitude by the respondents. This highlights why certain medical schools across the country offered their students a chance to graduate early and join the frontlines tackling the COVID-19 pandemic; medical students want to practice medicine, and they understand the need for dedicated providers.13 In addition, many previous studies have provided suggestions for engaging medical students throughout this pandemic,14,15 and it is reassuring to see medical students taking a stand for their communities and their education by staying engaged.16,17\n\nThough our sample population offers a unique perspective from each student, our study comes with certain limitations. Our response rate of 13% may reflect some non-responder bias as well as survey fatigue, as our survey was sent at a time when many students were experiencing distressing times. A more robust sample size might provide additional and more diverse viewpoints. Additionally, all respondents came from a single institution in the mid-Atlantic region. Students at institutions in regions that experienced very different case numbers, such as in the Midwest, South, or West, might have different experiences that alter their career trajectories. Lastly, there were a handful of respondents who left certain questions unanswered, leaving us with missing information for that student.\n\nDespite the challenges in distance-learning and career preparation that many are now experiencing, our survey study finds that students still have their minds set on achieving their pre-determined goals. We are hopeful that the hardships brought on by the COVID-19 pandemic have strengthened the next generation of physicians and that future patients benefit from their dedication.\n\n\nData availability\n\nFigshare: The Perceived Impact of the COVID-19 Pandemic on Medical Studentsâ Future Careers.\n\nDOI: https://doi.org/10.6084/m9.figshare.16879090.v17\n\nThis project contains the following underlying data:\n\n⢠Data file: Study survey questionnaire.\n\nRepository: The Perceived Impact of the COVID-19 Pandemic on Medical Studentsâ Future Careers.\n\nDOI: https://doi.org/10.6084/m9.figshare.16879087.v19\n\nThis project contains the following underlying data:\n\n⢠Data file: Student comments regarding step exams and other concerns.\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nAuthor contributions\n\nAll authors were involved in the conceptualization, investigation, methodology, project administration, supervision, validation, visualization, writing of the original draft, and reviewing and editing the subsequent drafts. HM and CV performed data curation and formal analysis. SMS was involved in funding acquisition, resources, and software.",
"appendix": "References\n\nGuidance on medical studentsâ participation in direct patient contact activities.Apr 2020. Reference Source\n\nVasavda C, Ho BK, Davison A: Socially Distant Medical Education in the Face of COVID-19. Med. Sci. Educ. Oct. 2020; 31: 231â233. Publisher Full Text\n\nUnited States Medical Licensing Examination|Announcements. (accessed Oct. 21, 2020). Reference Source\n\nGoldie JGS: Connectivism: A knowledge learning theory for the digital age?. Med. Teach. Oct. 2016; 38(10): 1064â1069. PubMed Abstract | Publisher Full Text\n\nView of Connectivism: Learning theory of the future or vestige of the past?|The International Review of Research in Open and Distributed Learning. (accessed Mar. 15, 2021).Reference Source\n\nYuen J, Xie F: Medical education during the COVID-19 pandemic: perspectives from UK trainees. Postgrad. Med. J. May 2020; 96: 432â433. PubMed Abstract | Publisher Full Text\n\nMahjoub H, Vasavda C, Bertram A, et al.: Supplemental Table 2 Survey Questions.docx. figshare. Dataset. 2021. Publisher Full Text\n\nBeck AT, Ward CH, Mendelson M, et al.: An Inventory for Measuring Depression. Arch. Gen. Psychiatry . Jun. 1961; 4(6): 561â571. Publisher Full Text\n\nMahjoub H, Vasavda C, Bertram A, et al.: Students Comments CC0.October 2021. Publisher Full Text\n\nMeo SA, Abukhalaf AA, Alomar AA, et al.: COVID-19 Pandemic: Impact of Quarantine on Medical Studentsâ Mental Wellbeing and Learning Behaviors. Pak. J. Med. Sci. May 2020; 36(COVID19-S4): S43âS48. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBabiker A, et al.: Health care professional development: Working as a team to improve patient care. Sudan. J. Paediatr. 2014; 14(2): 9â16. PubMed Abstract | Free Full Text\n\nByrnes YM, Civantos AM, Go BC, et al.: Effect of the COVID-19 pandemic on medical student career perceptions: a national survey study. Med. Educ. Online. 25(1). PubMed Abstract | Publisher Full Text | Free Full Text\n\nKelly M: Early Graduation Allows New Physicians to Join the Pandemic Fight Faster: In Light of the COVID-19 Crisis, Medical Schools Advance Their Students. Ann. Emerg. Med. 2020; 76(2): A15âA17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTariq EF, Sah PK, Malik A: The plight of COVID-19 pandemic on medical students and residency applicants. Ann. Med. Surg. (Lond). Dec. 2020; 60: 1â4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMiller DG, Pierson L, Doernberg S: The Role of Medical Students During the COVID-19 Pandemic. Ann. Intern. Med. Apr. 2020; 173: 145â146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohns Hopkins medical students help coronavirus patients and their families stay in touch - Baltimore Sun. (accessed Oct. 23, 2020).Reference Source\n\nDuring Pandemic, Medical Students Volunteer to Ensure No One Dies Alone. University of Michigan; (accessed Oct. 23, 2020).Reference Source"
}
|
[
{
"id": "118871",
"date": "10 Jan 2022",
"name": "Jason Yuen",
"expertise": [
"Reviewer Expertise My main clinical interests are in neurosurgery",
"particularly neuromodulation. In addition",
"I am interested in medical education and have completed a postgraduate certificate in medical education. I have also taught",
"examined and mentored medical students on multiple occasions."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of this paper conducted a single-institution, cross-sectional study of the perspectives of medical students on how the COVID pandemic impacted their education, well-being and future career. In particular, the authors found that, based on answers in the form of Likert scales from an online survey, medical students encountered difficulties in exploring different specialties. Yet, they continued to view medicine as a positive career choice.\nPros How COVID-19 impacts medical education remains highly topical given the evolving nature of the pandemic, although numerous articles on a similar topic have been published for the past 2 years. The questions of the survey were well-designed to capture the numerous ways COVID might affect a medical student.\nCons The study is heavily limited by the low response rate (13%). Therefore, the results may be confounded by selection bias, which the authors have rightly acknowledged.\nFurthermore, a significant proportion of the samples consist of students from the first 2 years, who were probably less affected in terms of specialty selection and doing external rotations.\nThis study was conducted within 1 year of the COVID pandemic and many of us would probably agree, there has been a significant amount of development, which makes the study less relevant. Yet, one can understand it does take time to process the data and prepare a manuscript (although it has taken over a year to do so in this case).\nI think statistically, the median would be more relevant to the mean as an average. I would suggest the authors providing that as well in case the results are skewed. As well as the average score, it may be useful to include percentages for certain questions, e.g., how many students answered âSomewhat agreeâ or âAgreeâ in the âI have considered quitting medical school during the pandemicâ question. It will be useful to include that in the supplemental information.\nIn addition, I feel the text is rather inconsistent when it comes to the description of data. For example, âWhen questioned about the adequacy of their medical education regarding epidemics, they felt neutral (3.49 ± 1.08).â and âThe students somewhat agreed that⊠it is their responsibility to volunteer during the pandemic (3.37 ± 1.12)â. A score of 3.49 is described as âneutralâ and yet a lower score of 3.37 is described as âsomewhat agreedâ.\nAs the authors pointed out, this is a single-institution study and may therefore not apply to other medical schools. Of note, in other countries, such as in the UK, newly-qualified doctors must rotate through a number of different specialties prior to applying for a specialty-specific residency program. Therefore, the issue in exploring different specialties during medical school becomes less important. It will be useful for the authors to discuss more recommendations on helping medical students, e.g., the introduction of virtual open days and rotations may help to keep them informed on different specialties.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9346",
"date": "03 Mar 2023",
"name": "Heba Mahjoub",
"role": "Author Response",
"response": "Reviewer 1, Jason Yuen, Department of Neurologic Surgery, Mayo Clinic, Rochester, MN, USA: Approved with Reservations Reviewer's comment: The authors of this paper conducted a single-institution, cross-sectional study of the perspectives of medical students on how the COVID pandemic impacted their education, well-being and future career. In particular, the authors found that, based on answers in the form of Likert scales from an online survey, medical students encountered difficulties in exploring different specialties. Yet, they continued to view medicine as a positive career choice. Pros How COVID-19 impacts medical education remains highly topical given the evolving nature of the pandemic, although numerous articles on a similar topic have been published for the past 2 years. The questions of the survey were well-designed to capture the numerous ways COVID might affect a medical student. Authors response: We thank you for your diligent review and positive reaction to our important work studying studentsâ perceived impact on their career trajectories during the COVID-19 pandemic. Reviewer's comment: Cons The study is heavily limited by the low response rate (13%). Therefore, the results may be confounded by selection bias, which the authors have rightly acknowledged. Furthermore, a significant proportion of the samples consist of students from the first 2 years, who were probably less affected in terms of specialty selection and doing external rotations. This study was conducted within 1 year of the COVID pandemic and many of us would probably agree, there has been a significant amount of development, which makes the study less relevant. Yet, one can understand it does take time to process the data and prepare a manuscript (although it has taken over a year to do so in this case). Authors response: The authors do acknowledge the low response rate, and we attribute survey fatigue as a major contributor given this time period was one where students were inundated with virtual surveys, especially since in-person research activities were shut-down to prevent spread of infection. However, we believe that first and second years were a crucial group to study, as these are the years where many students first encounter or begin to explore their preferred specialty. We also recognize the time since data collection and manuscript preparation, though this was heavily impacted by COVID-19 roadblocks such as illness and delayed reviewer identification. Reviewer's comment: I think statistically, the median would be more relevant to the mean as an average. I would suggest the authors providing that as well in case the results are skewed. As well as the average score, it may be useful to include percentages for certain questions, e.g., how many students answered âSomewhat agreeâ or âAgreeâ in the âI have considered quitting medical school during the pandemicâ question. It will be useful to include that in the supplemental information. Authors response: We checked for normality by analyzing kurtosis and skew using the Jarque-Barre test, and found the majority (16/23) of our questions met the criteria. Because of this, we do believe that the mean results are not skewed, but we have now also included median, 1st quartile, and 75th quartile in our final table to aid readers in visualizing the results. Reviewer's comment: In addition, I feel the text is rather inconsistent when it comes to the description of data. For example, âWhen questioned about the adequacy of their medical education regarding epidemics, they felt neutral (3.49 ± 1.08).â and âThe students somewhat agreed that⊠it is their responsibility to volunteer during the pandemic (3.37 ± 1.12)â. A score of 3.49 is described as âneutralâ and yet a lower score of 3.37 is described as âsomewhat agreedâ. Authors response: Thank you for bringing this to our attention. We have now added a line in the methods section clarifying which range value constitutes each variable: âAny value which rounded to the nearest 0.5 was included in the score above, for example a value of 3.49 was categorized as âsomewhat agreeâ.â  We also remedied 2 inconsistencies in the following lines: âWhen questioned about their medical education regarding epidemics, they somewhat agreed that their education was adequate (3.49 ± 1.08).â âThe students somewhat agreed that they view the field of medicine more positively since the onset of the COVID-19 pandemic (3.60 ± 1.09), but they felt neutral regarding their responsibility to volunteer during the pandemic (3.37 ± 1.12).â Reviewer's comment: As the authors pointed out, this is a single-institution study and may therefore not apply to other medical schools. Of note, in other countries, such as in the UK, newly-qualified doctors must rotate through a number of different specialties prior to applying for a specialty-specific residency program. Therefore, the issue in exploring different specialties during medical school becomes less important. It will be useful for the authors to discuss more recommendations on helping medical students, e.g., the introduction of virtual open days and rotations may help to keep them informed on different specialties. Authors response: Thank you for this unique perspective. It is true that these findings would be less relevant in other countries, such as the UK. We have included the following recommendation in the discussion section to be more helpful for students from all countries: âIn the future, students may benefit from earlier exposure to different specialties in a virtual platform. One example would be each specialty creating a âDay in the Lifeâ video at various institutions, which would expose students to the daily routine of each and help them make a decision about which experiences suited them best.â"
}
]
},
{
"id": "146011",
"date": "23 Aug 2022",
"name": "Aikaterini Dedeilia",
"expertise": [
"Reviewer Expertise medical education",
"surgical education",
"surgical oncology",
"general surgery",
"pediatric surgery"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present manuscript is a cross-sectional survey among medical students, on the effect of COVID-19 on their education and future career plans. The study is comprehensible and consistent, and adequately presents all the required information about the study process. I believe that it can be indexed, provided that the authors consider the following suggestions/comments and make any appropriate adjustments:\nIn the introduction, although it provides an adequate background for the study, I would suggest that more citations are added in the sentences that lack one and present an argument that needs verification (e.g.: \"Tasks that were once mundane have now become difficult or unsafe. Importantly, medical students have been uniquely affected.\" or \"Many medical students have...in the community.\")\n\nIn methods, the study design is adequately presented for any reader to understand. I would suggest that the authors add at the end of the first paragraph that continuous variables are presented as a mean followed by a standard deviation (mean ± SD), and that categorical variables as presented as n (%). Also, the phrase \"version XM\" is mentioned twice by accident in the same paragraph. Lastly, although it is highly important and proper of the authors to write the selection bias statement in the end of the methods, I would suggest that this limitation be included along with the rest of the limitations at the end of the discussion, along with the second limitation (about the single institution) that is repeated in the discussion.\n\nThe results are clear, legible, easily understandable and reproducible. I have no further comments or further improvements to make.\n\nThe discussion is related to the results, and the authors appropriately comment on the effect of COVID-19 on the career plans of medical students. I would suggest that more citations from similar studies are added in the discussion, in order to compare this study to others, or that some systemetic reviews that have studied this exact effect are added so that they further support the evidence from this study.\n\nLast but not least, if possible from the format of the journal guidelines, I would suggest that the last paragraph be presented as a separate conclusion (with its own subtitle), instead of as a part of a common \"discussion/conclusion\" section.\n\nIn conclusion, I have no further concerns or ethical reservations with regards to the present manuscript. I believe that it is a clear, reproducible, and technically sound study, that with some minor revisions can be indexed and add significant information to the literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9347",
"date": "03 Mar 2023",
"name": "Heba Mahjoub",
"role": "Author Response",
"response": "Reviewer 2, Aikaterini Dedeilia, Postdoctoral Research Fellow, Surgical Oncology Research Laboratories, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA; Research Fellow in Surgery, Harvard Medical School, Boston, MA, USA: Approved Reviewer's comment: The present manuscript is a cross-sectional survey among medical students, on the effect of COVID-19 on their education and future career plans. The study is comprehensible and consistent, and adequately presents all the required information about the study process. I believe that it can be indexed, provided that the authors consider the following suggestions/comments and make any appropriate adjustments: In the introduction, although it provides an adequate background for the study, I would suggest that more citations are added in the sentences that lack one and present an argument that needs verification (e.g.: \"Tasks that were once mundane have now become difficult or unsafe. Importantly, medical students have been uniquely affected.\" or \"Many medical students have...in the community.\") Authors response: Thank you for your encouraging feedback regarding the importance of these findings. We are eager to improve the work with your suggestions. We have added the following citations in our introduction section: Tasks that were once mundane, such as scheduling test dates, have now become difficult or unsafe.a Many medical students have also been unable to continue their scientific research and volunteer opportunities in the community.b a. Price, S. (2020). Testing Boundaries COVID-19 made the USMLE, Clerkships a Moving Target for Med Students. Tex. Med. 34:116. b. Pop AI, Mirel S, Florea M, Lotrean LM. The Impact of the COVID-19 Pandemic on Research and Volunteering Activities among Medical Students: A Cross-Sectional Study among Romanian and International Students from One Medical Faculty from Romania. Int J Environ Res Public Health. 2022;19(12):7477. doi:10.3390/ijerph19127477  Reviewer's comment: In methods, the study design is adequately presented for any reader to understand. I would suggest that the authors add at the end of the first paragraph that continuous variables are presented as a mean followed by a standard deviation (mean ± SD), and that categorical variables as presented as n (%). Also, the phrase \"version XM\" is mentioned twice by accident in the same paragraph. Lastly, although it is highly important and proper of the authors to write the selection bias statement in the end of the methods, I would suggest that this limitation be included along with the rest of the limitations at the end of the discussion, along with the second limitation (about the single institution) that is repeated in the discussion. Authors response: We appreciate your recommendation to include further description of how we represented our variables. We also have now removed the 2nd âversion XMâ that was erroneously included previously. Last, we removed the selection bias statement from the methods and inserted it in the discussion section along with other limitations. We have adjusted the methods as follows: âDescriptive statistics were calculated for all variables using Qualtrics ® version XM. Continuous variables are presented as a mean followed by a standard deviation (Mean ± SD), and categorical variables are presented as N (%).â The following line was inserted into the discussion section: âThe study is sensitive to selection bias for students that responded to and completed the survey, potentially limiting the generalizability of our findings from all students.â  Reviewer's comment: The results are clear, legible, easily understandable and reproducible. I have no further comments or further improvements to make. Authors response: Thank you for this positive feedback regarding the results!  Reviewer's comment: The discussion is related to the results, and the authors appropriately comment on the effect of COVID-19 on the career plans of medical students. I would suggest that more citations from similar studies are added in the discussion, in order to compare this study to others, or that some systemetic reviews that have studied this exact effect are added so that they further support the evidence from this study. Authors response: Thank you for the suggestion to include more citations in the discussion section. We have now included the following line and citations to support our findings: These findings align with several other studies conducted nationally and internationally.a,b,c a. Alsoufi A, Alsuyihili A, Msherghi A, et al. Impact of the COVID-19 pandemic on medical education: Medical studentsâ knowledge, attitudes, and practices regarding electronic learning. PLoS One. 2020;15(11):e0242905. doi:10.1371/journal.pone.0242905 b. Nicholas Pari Tekkis, Damir Rafi, Sam Brown, et al. (2022) The impact of the COVID-19 pandemic on UK medical education. A nationwide student survey, Medical Teacher, 44:5, 574-575, DOI: 10.1080/0142159X.2021.1962835 c. The perceived impact of the Covid-19 pandemic on medical student education and training â an international survey. BMC Med Educ. 2021;21:566. doi:10.1186/s12909-021-02983-3  Reviewer's comment: Last but not least, if possible from the format of the journal guidelines, I would suggest that the last paragraph be presented as a separate conclusion (with its own subtitle), instead of as a part of a common \"discussion/conclusion\" section. In conclusion, I have no further concerns or ethical reservations with regards to the present manuscript. I believe that it is a clear, reproducible, and technically sound study, that with some minor revisions can be indexed and add significant information to the literature. Authors response: Thank you for this suggestion, we have now separated the conclusion to stand on its own with its own subtitle. We appreciate your diligent work reviewing this study, and for your suggestions for improvement! We believe the edits we have made with your recommendations have made this a stronger manuscript."
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https://f1000research.com/articles/10-1211
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https://f1000research.com/articles/11-1561/v1
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22 Dec 22
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{
"type": "Research Article",
"title": "To evaluate and compare the effect of 17% EDTA, 10% citric acid, 7% maleic acid on the dentinal tubule penetration depth of bio ceramic root canal sealer using confocal laser scanning microscopy: an in vitro study",
"authors": [
"Shivangi Shekhar",
"P. Laxmish Mallya",
"Vasudev Ballal",
"Ramya Shenoy",
"Shivangi Shekhar",
"Vasudev Ballal",
"Ramya Shenoy"
],
"abstract": "Background: The main factors that affect the success of an endodontic infection are effective cleaning and shaping of the root canal system including complete disinfection by using chemical irrigating solutions and obturation with an endodontic sealer to provide a fluid-tight seal. Using rotary and hand instruments for shaping and cleaning the root canal along with irrigants produces a smear layer on the surface of root dentin affecting the penetration of the endodontic sealer into the dentinal tubules. This smear is difficult to remove with the use of only endodontic irrigants, hence, chelating agents were introduced in adjunct with irrigating solutions for irrigation protocol for effective removal of smear layer which effect the penetration of endodontic sealers into the dentinal tubules. Methods: 32 mandibular premolar teeth were used. The biomechanical preparation was done till Protaper F3 size. Irrigation was done with 2.5 mL sodium hypochlorite (NaOCl) solution after each instrumentation change for 1 min. Samples were then divided into 4 groups according to the irrigating solution used as the final rinse used with passive ultrasonic agitation. The groups were: Group I: 5 ml of saline, Group II: 5ml of 17% ethylenediaminetetraacetic acid, Group III: 5 ml of 10% citric acid, Group IV: 5 ml of 7% maleic acid each for one minute. All the canals were obturated with BioRoottmRCS with gutta-percha using the ultrasonic condensation technique. For staining the samples for Confocal LASER microscopy, BioRoottmRCS was mixed with Rhodamine B dye. Results: The maximum penetration of bio-ceramic sealer was observed in the coronal region. At the apical third, the maximum sealer penetration was seen with 7% maleic acid. Conclusions: Maximum sealer penetration was seen in the coronal section followed by the middle and apical section. Maximum sealer penetration was seen with 7 % maleic acid at the apical third.",
"keywords": [
"Biofilms",
"Citric Acid",
"EDTA",
"Maleic Acid",
"Smear Layer."
],
"content": "Introduction\n\nThe main purpose of an endodontic treatment is to eradicate the microorganisms, debris and necrotic tissue prevailing in the root canals and to provide effective cleaning and decontamination of the root canal system to avoid its re-contamination.1 Chemomechanical preparation plays a significant role in the endodontic treatment.2 The root canal at the apical region has a complex structure containing cul de sacs, lateral and accessary canals which makes the debridement of infected tissue from this area a challenging task.3 Even after chemomechanical preparation using hand or rotary instruments, an average amount of 30-35% root canal area is left untouched and un-instrumented. Irrigating solutions do not reach this area and hence no proper cleaning of such areas leads to re-contamination and failure of the treatment.4 The process of root canal cleaning and its shaping is established by biomechanical preparation and extensive irrigation using endodontic irrigants.5 Chemo mechanical preparation using hand or rotary instruments produces a smear layer which is an uneven amorphous granular layer present on the root dentin containing both inorganic and organic contents like necrotic debris, odontoblastic processes, pulp tissue and microorganisms with their metabolic products. Although there is debate if the smear layer should be removed or kept, a recent meta-analysis and systematic review concluded that removing the smear layer improved the root canal system's fluid tight seal.6 The smear layer has been demonstrated to be infected and protects the microorganisms present in the dentinal tubule and also inhibits the penetration of endodontic sealers and intracanal medicaments into the dentinal tubules which leads to some bacteria being left behind in the dentinal tubules which might later cause re-infection.7\n\nIrrigating solutions for endodontic treatment play a significant part in the success of the treatment. The process of instrumentation along with administration of the root canal irrigants, facilitates the elimination of microorganisms, smear layer, tissue fragments and necrotic debris present in the root canal completely by the flushing mechanism of action of the irrigants.8 These irrigants have a benefit of reaching the accessory features and complex area such as lateral and accessory canals, and cul-de-sacs which are generally hard to access. Agitation and activation of the irrigants through lasers, sonics and ultrasonics are additional sources which help to improve the smear layer removal along with irrigants used for canal irrigation.9,10 Passive ultrasonic irrigation (PUI) employs ultrasonic wave energy that is transmitted from a tip or file to the irrigant and produces cleaner canals.11 To eradicate the smear layer, endodontists have used various chelating chemicals such as ethylenediamineactetic acid (EDTA), phosphoric acid, maleic acid (MA), citric acid (CA) and etidronic acid. The use of demineralizing agents along with sodium hypochlorite irrigation has been suggested for active removal of smear layer for successful endodontic treatment. Ballal et al.12 in their research concluded that that final irrigation during biomechanical preparation with 7% MA for one min was more active in eradicating the smear layer when compared to 17% EDTA at the apical region of the root canal. Demirel et al. in their study revealed that irrigation with 6 % citric acid was more efficient compared to 10% EDTA for eradicating smear layer present in root canal dentin.13\n\nSince complete elimination of microorganisms from the endodontic space is unlikely, the antimicrobial activity of root canal sealers may help to remove residual microorganisms which remain unaffected by chemo mechanical preparation and irrigating solutions used for the root canal treatment. Vibha et al. conducted a study to assess the depth of penetration of sealer into dentinal tubules at the apical, middle and coronal third of root canals, and concluded that in the apical sections of CA and EDTA showed comparable sealer penetration and maximum sealer penetration was seen at the coronal portion.14 To date, no comparative studies have been published that compare the smear layer removal and the penetration depth of Bio-ceramic sealer into the dentinal tubules present in the root canal.\n\nThe goal of this in vitro study is to use a Confocal LASER scanning microscope to compare the effect of saline, EDTA, MA, and CA solutions on removal of smear layer and to measure depth of penetration of Bio ceramic sealer (BioRoottmRCS) into the dentinal tubules at the coronal, middle and apical third regions of the root canal. The null hypothesis tested is that there is no significant difference in smear layer and sealer penetration among saline, 7% MA, 10% CA and 17% EDTA when used as final irrigating solution.\n\n\nMethods\n\nThe ethical committee clearance was attained from the institutional (Manipal College of Dental Sciences) ethical committee (reference number-19083 (18/09/2019)).\n\nSample size was based on Meadâs resource equation,15 where the total number of samples in the study was based on blocking component, treatment component and error component. A total of 32 samples and per group 8 samples were included. 32 mandibular premolar teeth extracted for orthodontic purpose with straight roots, single canal and no resorption or caries were used. As these are routinely extracted teeth, they were easily obtained from the orthodontic clinic. Hence patient consent was not required.\n\nThe teeth used were kept in a solution of 0.2% sodium azide (Sigma Chemical Co, St Louis, MO) at 4°C. The occurrence of one canal in the teeth was confirmed radiographically on three films angulated at different angles. The teeth were decoronated at cementoenamel junction (CEJ) with a 0.1mm diamond disc to standardize the length of the root to 15 mm from the anatomic apex.15 The working length (WL) of each tooth was calculated by introducing a 15 K file (Mani Inc, Tochigi Ken, Japan) into each canal until it was seen at the root apex and subtracting 1 mm from this point and then later confirmed with radiographs. Sizes 2 and 3 of Gates Glidden drill (Mani Inc, Tochigi Ken, Japan) were used to enlarge the coronal region for easier access to the middle and apical regions of the root canal. The apical region of each canal was enlarged till ISO 40 K file (Mani Inc, Tochigi Ken, Japan).15 The chemo mechanical preparation was done using ProTaper files till size F3 (Dentsply Protaper Universal) by applying the Crown Down technique.16\n\nIrrigation of the root canal was performed with 2.5 mL NaOCl (Vishal Dentocare Pvt. Ltd, INDIA) solution after each instrumentation change for one minute.17\n\nSamples were further divided into four groups with eight samples according to final irrigating solution used with passive ultrasonic agitation.\n\nThe groups were:\n\nGroup I: 5 ml of 0.9% physiological saline for 1 minute\n\nGroup II: 5ml of 17% EDTA for 1 minute\n\nGroup III: 5 ml of 10% CA for one minute for 1 minute\n\nGroup IV: 5 ml of 7% MA for 1 minute.\n\nIrrigation was done using 2-ml disposable plastic syringe with a side vented 27-gauge needle (Dispovan, Mumbai, Maharashtra, India) which was inserted 1 mm short of the WL. Passive ultrasonic agitation was done after the irrigation with the irrigating solutions. For passive ultrasonic agitation, a side vented 30-guage was used. Ultrasonic file (Irrisafe, Satelec, Aceton Group, Merignac cedex, France) of size 20, 2% taper was introduced into the canal 1mm short of the apex. Ultrasonic activation was carried out for one minute with a power of 3 using an ultrasonic unit (Suprasson P5 Booster, Satelec). It was repeated three times to maintain a total span for three minutes. After the final irrigation with the solutions the root canals were dried up using paper points. BioRoottm RCS, a bio-ceramic sealer (Septodont) was used for obturation. The sealer was applied to the canal walls using a lentino spiral and obturation using gutta percha was done using the lateral condensation technique.17\n\nFor fluorescence under confocal microscopy, Rhodamine B dye (Loba Chemie Pvt. Ltd.) was mixed with BioRoottm RCS sealer. After obturation the teeth were stored in a humidifier with 100% humidity for twenty-four hours which allowed the sealer to set in the presence of moisture. After 24 hours, all the sample were removed from the humidifier and were segmented at distances of 2 mm, 5 mm and 8 mm from the root apex equivalent to apical, middle and coronal regions of each tooth.15 All the specimens were sectioned using a diamond disc at 90° to the long axis of the tooth with a straight handpiece and micromotor unit (Confident Dental Equipments Ltd.).\n\nThe sections were kept flat at 2mm thickness each for ease of mounting onto the glass slides for Confocal Scanning Electron Microscopy (CSLM). The specimens were kept on the glass slides provided and were then examined under confocal LASER scanning microscope. The confocal LASER microscope used was DPSS model DMi8. All the sections of the root specimen were scanned at 10Ã. The excitation was kept at 561 nm to collect the emission produced at 585-682 nm mode. Images obtained on the computer were then processed for background noise reduction (Leica Application Suite X, version 3.5.7.23225). Depth of penetration of sealer was recorded for each part and mean value of each section was calculated. Digital ruler inbuilt in the software (LAS-AF, Leica) was used for measuring the depth of sealer penetration.18\n\nStatistical product and service solutions, version 20.0 (SPSS Inc., Chicago, IL, USA) was used in analysis. Statistical analysis was performed by using the one-way ANOVA. The post hoc Tamhanes test was applied for comparisons between the groups (intergroup analysis) and within the groups (intragroup analysis). The level of significance (P) was kept at 5%.\n\n\nResults\n\nThe result of the present study concluded that the maximum depth of Bio-ceramic sealer penetration of all the groups were seen at the coronal third followed by middle and apical third of the root specimens examined (Table 1).\n\nThe one way ANOVA shows a statistically significant difference between the groups and within the groups. Results showed statistical significance with the df 2; F value 2, P value- .000 (Table 2). The results showed the saline group had a significantly lower depth of sealer penetration compared to the other groups in all the three sections of the root specimen (P < .05).\n\nThe results of the inter group analysis demonstrated that there was a significant statical difference seen in the depth of penetration of sealer between all irrigating solutions at the coronal, middle and apical third of the root specimen (P < .05). No statistically significant difference in the depth of bio- ceramic sealer penetration between EDTA, CA, MA at coronal third was observed. At the middle and apical regions, a statistically significant difference was seen in the depth of penetration of sealer between EDTA and CA and MA (Table 3).\n\nFor intra group analysis, the post-hoc Tamhanes test results showed that there was a significant difference (P < .05) among all final irrigating solutions at different sections of the root specimen (Table 4).\n\nFor EDTA, the maximum sealer penetration depth was seen at coronal third which had no statistically significant difference with CA and MA, suggesting that all the three irrigating solutions were similarly effective in removal of smear layer at coronal third.\n\nAt the middle and apical third, a statistically significant difference was seen between EDTA and CA and MA suggesting that CA and MA were more active than EDTA in removal of smear layer at middle third, and hence greater penetration of Bio-ceramic sealer into the dentinal tubules were seen with CA and MA.\n\nNo statistical difference was seen between CA and MA at middle third and apical third suggesting that both were similarly effective in removal of smear layer hence influenced the penetration of bio ceramic sealer in the dentinal tubules.\n\nFigure 1 displays a representation of Confocal LASER scanning microscopic images from the Citric acid group (C: Coronal, M: Middle, A: Apical). Figure 2 displays a representation of Confocal LASER scanning microscopic images from the Saline group. Figure 3 displays a representation of Confocal LASER scanning microscopic images from the Maleic acid group, and Figure 4 displays a representation of Confocal LASER scanning microscopic images from the Eythenediamine tetracetic acid group.\n\n(C: Coronal, M: Middle, A: Apical).\n\n(C: coronal, M: Middle, A: Apical).\n\n(C: coronal, M: Middle, A: Apical).\n\n(C: coronal, M: Middle, A: Apical).\n\n\nDiscussion\n\nThe current study was done to evaluate the efficacy of 17 % EDTA, 10% CA, 7% MA and 0.9% physiological saline in the depth of penetration of bio ceramic sealer into the dentinal tubules of different sections of the root specimens. To date, not much evidence has been published for penetration depth of Bio-ceramic sealer into the dentinal tubules of root canal dentin. Hence, this study included a Bio-ceramic sealer as they set even in the presence of moisture not affecting its properties.\n\nThe literature shows that most irrigants used for irrigation along with biomechanical preparation are not effectively active against smear layer at all the sections of the root specimen, particularly at the apical region, which is vital in determining the prognosis of an endodontic treatment. The results of this study revealed that both 7% MA and 10% CA were equally effective in removing smear layer at middle and apical third. To facilitate a satisfactory cleaning and infiltration of irrigants into the dentinal tubules, the apical region of each specimen was enlarged until size 40 no K file. This was done in agreement with other studies that have concluded that a greater apical preparation allows a greater decrease in residual bacteria and aids in better smear layer removal as compared with smaller preparation.19,20 Passive ultrasonic agitation was done to produce acoustic streaming to eliminate debris from the canal space and leave behind cleaner canals. Passive ultrasonic agitation when used in combination with conventional syringe irrigation produces clear canals. This is in agreement with earlier studies.11 The most important factors that affect the action of the irrigating solution are the concentration of the irrigant used and its contact time with the root canal dentin. The exact optimum contact time needed for the irrigant to be held in reserve in root canals for effective removal of smear layer is unclear. Studies have shown that EDTA reasonably removes the smear layer in one min, but it also produced extreme erosion of intertubular and peritubular dentin when it was applied for duration of more than 10 mins.21 Ballal et al.12 in their study concluded that final irrigation for one min with 7% MA was effective at the apical third in smear layer removal. Therefore, the irrigation time of one min was kept for the irrigants used in this particular study.\n\nThe results of the present study revealed that 7% MA and 10% CA had greater depth of bio ceramic sealer penetration into the dentinal tubules suggesting they had a better capability for removing smear layer when compared to 17% EDTA in the apical and middle third regions of the root specimen. No statistically significant difference was seen in the depth of the Bio-ceramic sealer penetration between 7% MA and 10% CA at the middle and apical third suggesting that at apical third both the irrigants were equally effective in eliminating the smear layer. This result is in accordance with other studies.22,23\n\nEDTA is a chelating solution which aids in the elimination of smear layer by facilitating the elimination of microorganisms present in the canal space, thereby improving the anti-microbial effect of disinfecting agents in further deeper layers of dentin. The consequence of EDTA on dentin totally depends on the total time it is in contact with dentin and also on the concentration of EDTA solution. Several previous studies resolved that irrigation in the endodontic treatment with EDTA appears to be a promising endodontic tool.24,25 However, in our present study, the depth of sealer penetration into dentinal tubules with EDTA was lower at the middle and the apical third when compared to 7% MA and 10% CA suggesting it was less effective in removing the smear layer at these areas. Even after larger apical preparation, EDTA was less effective when compared with MA and CA to eliminate smear layer successfully. The reason could be the higher surface tension of 17% EDTA. A previous study concluded that surface tension of 7% MA is lower compared to 17% EDTA.26 Since EDTA is extremely active at a neutral pH and a decrease in pH over time is seen. Hence, its efficacy with time decreases as decrease in pH causes decrease in its effectiveness.26 Paque et al.21 concluded that EDTA does not have a noticeable action in apical region of the root canal since the dentin present in this region is sclerosed. Goldberg et al. concluded that optimal results are obtained only after an application time of minimum 15 mins with EDTA, but in this study the application time was only one minute hence EDTA was not as effective removing smear at the apical third.27\n\nResearch done by Banode et al.28 presented that the final irrigation with citric acid showed better results compared to EDTA in removing the smear layer from the canal space. In this present study, 10% citric acid solution was used because of its biocompatibility in addition to its ability to eliminate microorganisms, infected tissue and inorganic smear layer present in the root canal dentin. This is in accordance with study done by Malheiros29 which concluded that 10â25% citric acid has better biocompatibility compared to 17% EDTA. The citric acid used was effective in all sections of the tooth. This result is in accordance with Schaferâs declaration that 1â40% citric acid can be used as an irrigant for endodontic treatment as citric acid is an organic acid which has the ability to demineralize the sclerosed dentin present at the apical region of the root canal.30\n\nMA produces a better demineralizing effect in a briefer period of time because itâs highly acidic at lower pH within a shorter period of time as it has a greater demineralizing effect when compared to EDTA. As stated above, the dentin present at the apical region of the root canal is greatly sclerosed. The surface tension of 7% maleic acid is less when compared to EDTA hence its acidity doesnât increase, and it remains highly acidic at low ph. The maleic acid as stated above has the ability of eliminating the smear layer and demineralising inter and peri tubular dentin is because of its low pH of 1.05. Hence, the maleic acid used in this study was efficient in eliminating smear layer from all the sections of the root specimen. There was no statistically significant difference seen between 7% MA, 10% CA and 17% EDTA at the coronal third. The results showed no statistical significance between 7% MA and 10% CA at apical and middle third, concluding that both were better than 17% EDTA in eradicating smear layer at those sections of the root specimen. For the above reasons, better smear layer removal and depth of penetration of Bioceramic sealer was seen with MA and CA at the apical and middle sections significant with the other groups. All the specimens analysed for the control group were heavily smeared in all the sections (coronal, middle and apical third) of the root specimen.30\n\nThe maximum penetration with Bio-ceramic sealer was seen in the coronal section of the root specimen for all the tested irrigants solutions concluding that better smear layer removal was seen at the coronal third. The result is in agreement with the previous study done by Kara et al. which determined that the supreme penetration depth of an endodontic sealer was seen in the coronal region when compared to the apical region and various other studies.31,32 This may be due to larger diameter at the coronal and middle third areas, allowing an improved movement of the irrigating solutions.13 Difficulty in cleaning the apical most portion of the root canal can be explained by decrease in the diameter of the root canal at the apex, which decreases the access of irrigants at the apex which consequently results in reduction of its flow. These features donât allow the irrigants to reach the entire working length and hence the apical area remains uncleaned. Irfan et al. concluded that features that could have an impact in the cleaning of the apical area of the root canals are the complex anatomical configuration seen at the apex, limited space available, low permeability and difficulty of access.33\n\nThe Bio-ceramic sealer used for the study was BioRoot RCS. It is a mineral based permanent sealer used for obturating the root canal. It has an excellent adhesion to dentin and also to the gutta percha cones. It is hydrophilic in nature and continues its adhesion even in the presence of moisture. As it was an in vitro study, the exact in vivo conditions could not be simulated. Confocal LASER microscopy is a well-known light microscopical technique for imaging fluorescently dyed samples with significant three-dimensional structure. It has the ability to capture images at dissimilar depths in a model to enable to reform the three-dimensional structures within an object. Samples used are generally treated with fluorescent dyes to make objects visible. Confocal microscopy delivers the capacity for non-invasive, direct, serial optical sectioning of living specimens in lateral resolution.34,35\n\nFurther in vitro studies are required with to check the efficiency of 10% citric acid in removal of smear layer at different sections of the tooth. Further research is obligatory to evaluate the sealer penetration depth of endodontic sealers with other biocompatible chelating irrigants gaining importance lately.\n\n\nConclusion\n\nMaximum sealer penetration was seen in the coronal section followed by middle and apical sections of the root specimen. 17% EDTA, 10% CA, 7% MA were equally effective in sealer penetration at coronal third. However, in middle and apical section, 10% CA and 7% MA showed better efficacy in removal of smear layer compared to 17% EDTA, thereby showing more sealer penetration. Saline showed the least penetration of sealer in the dentinal tubules.",
"appendix": "Data availability\n\nFigshare: Raw Data, https://doi.org/10.6084/m9.figshare.21280152.v7. 36\n\nThis project contains the following underlying data:\n\n- RAW DATA OF SPECIMENS EXAMINED.xlsx (raw data as values of depth of sealer penetration into the dentinal tubules measured by an in-built ruler in mm in Confocal Laser Scanning Microscopy)\n\n- JPEG images of coronal, middle and apical third sections of the root specimen.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nSiqueira JF Jr: Aetiology of root canal treatment failure: why well-treated teeth can fail. Int. Endod. J. 2001 Jan; 34(1): 1â10. PubMed Abstract | Publisher Full Text\n\nStasinopoulos E: The importance of the chemomechanical preparation of the root canals in endodontic treatment: preliminary report. Odontiatriki. 1972 Mar-Apr; 2: 138â143. PubMed Abstract\n\nZolty G: The prevalence and significance of sealing accessory and lateral canals: a literature review. SADJ. 2001 Sep; 56(9): 417â424. PubMed Abstract\n\nAung NPS, Watanabe S, Okiji T: Er:YAG Laser-Activated Irrigation in Comparison with Different Irrigation Systems for Cleaning the Apical Root Canal Area Beyond Ledge. Photobiomodul. Photomed. Laser Surg. 2021 Dec; 39(12): 759â765. PubMed Abstract | Publisher Full Text\n\nde Macedo LMD , Silva-Sousa YTC, Olivato Junior O, et al.: Different biomechanical preparation protocols on the penetration and bond strength of the filling material to dentin. Braz. Dent. J. 2021 Sep-Oct; 32(5): 12â22. PubMed Abstract | Publisher Full Text\n\nShahi S, Yavari HR, Rahimi S, et al.: A comparative scanning electron microscopic study of the effect of three different rotary instruments on smear layer formation. J. Oral Sci. 2009 Mar; 51(1): 55â60. 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Conserv. Dent. 2019 Mar; 22(2): 149â154. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPlotino G, Cortese T, Grande NM, et al.: New technologies to improve root canal disinfection. Braz. Dent. J. 2016 Jan; 27: 3â8. PubMed Abstract | Publisher Full Text\n\nBallal NV, Kandian S, Mala K, et al.: Comparison of the efficacy of maleic acid and ethylenediaminetetraacetic acid in smear layer removal from instrumented human root canal: A scanning electron microscopic study. J. Endod. 2009; 35: 1573â1576. PubMed Abstract | Publisher Full Text\n\nDemirel A, YÃŒksel BN, Ziya M, et al.: The effect of different irrigation protocols on smear layer removal in root canals of primary teeth: a SEM study. Acta Odontol. Scand. 2019 Jul; 77(5): 380â385. PubMed Abstract | Publisher Full Text\n\nLoganathan M: Comparative evaluation of penetration depth and push-out bond strength of C points and gutta percha using bioceramic & AH plus sealer: An Invitro study.(Doctoral dissertation, KSR Institute of Dental Science and Research, Tiruchengode).\n\nTuncer AK, Tuncer S: Effect of different final irrigation solutions on dentinal tubule penetration depth and percentage of root canal sealer. J. Endod. 2012 Jun 1; 38(6): 860â863. PubMed Abstract | Publisher Full Text\n\nUsman N, Baumgartner JC, Marshall JG: Influence of instrument size on root canal debridement. J. Endod. 2004 Feb 1; 30(2): 110â112. PubMed Abstract | Publisher Full Text\n\nCarver K, Nusstein J, Reader A, et al.: In vivo antibacterial efficacy of ultrasound after hand and rotary instrumentation in human mandibular molars. J. Endod. 2007 Sep 1; 33(9): 1038â1043. PubMed Abstract | Publisher Full Text\n\nvan der Sluis LW , Versluis M, Wu MK, et al.: Passive ultrasonic irrigation of the root canal: a review of the literature. Int. Endod. J. 2007 Jun; 40(6): 415â426. PubMed Abstract | Publisher Full Text\n\nMancini M, Cerroni L, Iorio L, et al.: Smear layer removal and canal cleanliness using different irrigation systems (EndoActivator, EndoVac, and passive ultrasonic irrigation): field emission scanning electron microscopic evaluation in an in vitro study. J. Endod. 2013 Nov; 39(11): 1456â1460. PubMed Abstract | Publisher Full Text\n\nAmin K, Masoodi A, Nabi S, et al.: Effect of diode laser and ultrasonics with and without ethylenediaminetetraacetic acid on smear layer removal from the root canals: A scanning electron microscope study. J. Conserv. Dent. 2016 Sep-Oct; 19(5): 424â427. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAttur K, Joy MT, Karim R, et al.: Comparative analysis of endodontic smear layer removal efficacy of 17% ethylenediaminetetraacetic acid, 7% maleic acid, and 2% chlorhexidine using scanning electron microscope: An in vitro study. J. Int. Soc. Prev. Community Dent. 2016; 6: S160âS165. PubMed Abstract | Publisher Full Text\n\nSjogren U, Hagglund B, Sundqvist G, et al.: Factors affecting the long-term results of endodontic treatment. J. Endod. 1990 Oct; 16(10): 498â504. Publisher Full Text\n\nOliveira KV, Silva BMD, Leonardi DP, et al.: Effectiveness of different final irrigation techniques and placement of endodontic sealer into dentinal tubules. Braz. Oral Res. 2017 Dec 18; 31: e114. Publisher Full Text\n\nGonçalves LS, Rodrigues RC, Andrade Junior CV, et al.: The Effect of Sodium Hypochlorite and Chlorhexidine as Irrigant Solutions for Root Canal Disinfection: A Systematic Review of Clinical Trials. J. Endod. 2016 Apr; 42(4): 527â532. PubMed Abstract | Publisher Full Text\n\nRuksakiet K, Hanák L, Farkas N, et al.: Antimicrobial Efficacy of Chlorhexidine and Sodium Hypochlorite in Root Canal Disinfection: A Systematic Review and Meta-analysis of Randomized Controlled Trials. J. Endod. 2020 Aug; 46(8): 1032â1041.e7. PubMed Abstract | Publisher Full Text\n\nHaapasalo M, Shen Y, Wang Z, et al.: Irrigation in endodontics. Br. Dent. J. 2014 Mar; 216(6): 299â303. Publisher Full Text\n\nGoldberg SN: Radiofrequency tumor ablation: principles and techniques. Multi-Treatment Modalities of Liver Tumours. 2002: 87â118. Publisher Full Text\n\nBanode AM, Gade V, Patil S, et al.: Comparative scanning electron microscopy evaluation of smear layer removal with 17% ethylenediaminetetraacetic acid, 10% citric acid and newer irrigant QMix: in vitro study. Indian J. Oral Health Res. 2015 Jul 1; 1(2): 56. Publisher Full Text\n\nMalheiros CF, Marques MM, Gavini G: In vitro evaluation of the cytotoxic effects of acid solutions used as canal irrigants. J. Endod. 2005 Oct 1; 31(10): 746â748. PubMed Abstract | Publisher Full Text\n\nSchÀfer E: Irrigation of the root canal. Endodontic Practice Today. 2007 Mar 1; 1(1).\n\nFarouz R, Delzangles B, Laurent E, et al.: PremiÚre partie: Pourquoi faut-il l'éliminer? [The endodontic smear layer. 1. Why should it be removed?]. Rev. Odontostomatol. (Paris). 1988 Mar-Apr; 17(2): 107â115.\n\nCergneux M, Ciucchi B, Dietschi JM, et al.: Etude de l'influence de la smear layer sur l'étanchéité de l'obturation canalaire [The effect of the smear layer on the impenetrability of the root canal obturation]. Inf. Dent. 1986 May 1; 68(18): 1699â1702. PubMed Abstract\n\nIrfan M, Suvarna N, Shetty H, et al.: The effect of 10% citric acid, 7% maleic acid & MTAD on intracanal smear layer removal A SEM study. Endodontology. 2013; 25: 30â36. Publisher Full Text\n\nRaghavendra SS, Jadhav GR, Gathani KM, et al.: Bioceramics in endodontics - a review. J. Istanb. Univ. Fac. Dent. 2017 Dec 2; 51(3 Suppl 1): S128âS137. Publisher Full Text\n\nPaddock SW, Eliceiri KW: Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques. Methods Mol. Biol. 2014; 1075: 9â47. PubMed Abstract | Publisher Full Text\n\nMallya L, Shekhar S:Raw Data. figshare. Dataset.2022. Publisher Full Text"
}
|
[
{
"id": "158693",
"date": "07 Feb 2023",
"name": "Saravana Karthikeyan Balasubramanian",
"expertise": [
"Reviewer Expertise Dentistry",
"Endodontics",
"Tissue Engineering"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for rendering an opportunity to review the current article for the reputed journal, F1000Research. This interesting clinically significant study compared the effect of various root canal chelating agents by ultrasonic agitation on the dentinal tubule penetration of BioRoottmRCS , a bioceramic root canal sealer. However, this manuscript requires minor modifications before indexing. Please find my comments attached herewith:\nAbstract:\n\nBackground, Line 5: The authors need to mention as smear layer rather smear. Also, please state the aim/rationale of the study.\n\nMethods: Please mention as 32 decoronated mandibular premolars.\n\nResults: The authors are suggested to briefly address the mean penetration values of the BioRoot RCS following the use of various irrigants at the coronal, middle and apical thirds. If not, please mention the mean values of significant groups atleast. The authors also need to highlight the type of statistical tests and the significance level employed in the study.\n\nConclusions: The authors can suggest some recommendations as well about the use of maleic acid when compared to other irrigants.\n\nManuscript:\nIntroduction: Please highlight the drawbacks of 17% EDTA, the gold standard chelating agent. This further validates the rationale for the use of alternative chelating agents like CA and MA.\n\nDiscussion: Though, authors had well justified their results, it is recommended that the contraindicatory studies that favor the use of EDTA also need to be highlighted.\n\nAcceptance/Rejection of Null hypothesis based on the current study results need to be done.\n\nLimitations of the study can be mentioned.\n\nThe authors had mentioned that future in vitro studies are required pertaining to the use of 10% CA alone. They can also mention about the detailed exploration of the comparative studies between the three chelating agents as well.\n\nAnd the scope of the study that includes future ex vivo studies or in vivo animal studies can be addressed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "9371",
"date": "22 Feb 2023",
"name": "P. Laxmish Mallya",
"role": "Author Response",
"response": "Noted Sir. Thank you for the corrections. I will try to upload a new version of the manuscript with all the corrections. Thank you Sir."
}
]
},
{
"id": "160806",
"date": "13 Feb 2023",
"name": "Amber Ather",
"expertise": [
"Reviewer Expertise Endodontics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for giving me an opportunity to review this paper by Shekhar et al. reporting on an important topic in the field of endodontics. This manuscript is focused on comparing different irrigant solutions and its effect on penetration depth of sealer.\nPlease find my suggestions and concerns with manuscript below:\n\nThere are at least a few other papers published in literature comparing irrigants and its effect on the penetration of bioceramic sealer in human teeth. The literature search needs to redone and should include all the relevant data. Some of the relevant papers are as follows:\nAlim Uysal BA, Kotan G, Guneser MB, Dincer AN, Senturk H, Rafiqi AM. Investigation of the effect of different chelation solutions on penetration of resin-based and bioceramic sealers with a novel method. Microsc Res Tech. 2021;84(7):1571-1576. doi:10.1002/jemt.23717\nStudy has looked at various chelating solutions including Maleic acid on penetration of bioceramic sealer. Please include this study in the introduction and/or discussion section.\n\nEskander M, Genena S, Zaazou A, Moussa S. Effect of phytic acid and ethylenediaminetetraacetic acid on penetration depth of bioceramic and resin sealers. Aust Endod J. 2021;47(3):506-511. doi:10.1111/aej.12513\nThis study is focused on the same so authors are requested to include this as well.\n\nGawdat SI, Bedier MM. Influence of dual rinse irrigation on dentinal penetration of a bioceramic root canal sealer: A Conofocal microscopic Analysis. Aust Endod J. 2022;48(3):481-486. doi:10.1111/aej.12599\n\nThere are several errors with regard to references, which needs to be corrected.\nE.g. authors mention reference #6 as a systematic review and meta-analysis in their manuscript; however, itâs an in vitro study. The authors are requested to refrain from using indirect references and cite the original paper. Similarly, in âIntroductionâ section, paragraph 3rd, line 3, authors have stated an author âVibha et alâ and have ended the sentence with reference #14. Reference #14 corresponds to âLoganathan Mâ in the bibliography. These are just few of the referencing and bibliography errors.\n\nIn âMaterials and methodsâ, for root canal preparation, the authors mention the use of size 40 hand file to Working length and then use of Protaper F3 for final preparation. If apical prep was already done till size 40/02, then Protaper F3 corresponding to size (30/09 variable taper) will not alter the apical prep. The authors are requested to clarify the instrumentation sequence and the final Master apical file.\n\nIn Discussion section, the literature published on bioceramic sealer penetration needs to be discussed and compared with the studyâs findings.\n\nPlease include the limitations of the study.\n\nThe conclusion does not include the focus on âbioceramic sealerâ. Please rephrase the conclusion, as according to authors this is what makes their research novel.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "9372",
"date": "22 Feb 2023",
"name": "P. Laxmish Mallya",
"role": "Author Response",
"response": "Noted Sir. Thank you for the corrections. I will try to upload a new version of the manuscript with all the corrections. Thank you Sir."
}
]
}
] | 1
|
https://f1000research.com/articles/11-1561
|
https://f1000research.com/articles/12-209/v1
|
24 Feb 23
|
{
"type": "Study Protocol",
"title": "State-of-the-art of invasive Group A Streptococcus infection in children: protocol for a scoping review of the literature with a focus on predictors of invasive infection",
"authors": [
"Francesco Mariani",
"Laura Martino",
"Carolina Gentili",
"Valentina Pulcinelli",
"Piero Valentini",
"Danilo Buonsenso",
"Francesco Mariani",
"Laura Martino",
"Carolina Gentili",
"Valentina Pulcinelli",
"Piero Valentini"
],
"abstract": "Background: Invasive group A streptococcus infection (iGAS) is a serious, sometimes life-threatening condition, with high case fatality rates and high morbidity whose incidence is greatly increased in the last years. Despite the increasing importance and frequency of this condition, at the best of our knowledge, no previous reviews have been published focusing on the risk factors for the development of this condition and its early clinical features. This paper reports the study protocol for a scoping review that aims to analyze the early signs and clinical features of invasive group A streptococcus disease in children, to recognize the prodromal stage of the disease. Methods: Comprehensive research combining the terms pediatric and invasive group A streptococcus infection has been performed on PubMed and SCOPUS to identify potential eligible studies. The search strategy for PubMed will be available in this paper. Two reviewers will screen first the abstract and subsequently the full text to identify eligible articles according to the predefined inclusion criteria. Divergences between the reviewers will be resolved by discussion (with a third author if necessary). Two review authors will extract data independently, everyone on a different Excel spreadsheet. Each researcher will be blinded to the decision of the other researcher. When the process will be completed, in case of discordance, any disagreement will be identified and resolved through discussion (with a third author if necessary). Dissemination: The findings of this review will be published in a peer-reviewed journal.",
"keywords": [
"streptococcus",
"group A streptococcus",
"iGAS"
],
"content": "Introduction\n\nStreptococcus Pyogenes (Group A Streptococcus, GAS) can cause different infections, ranging from minor illnesses such as pharyngitis and superficial skin infections to severe diseases. An invasive disease is defined as the isolation of GAS from a normally sterile site of the body and it occurs when bacteria spreads throughout the bloodstream, the cerebrospinal fluid, the lungs and soft tissue. Group A Streptococcus Invasive infection (iGAS) is a serious, sometimes life-threatening condition, with high case fatality rates and high morbidity.\n\nAccording to CDC Active Bacterial Core surveillance reports, overall invasive GAS incidence increased every year from 2012 to 2019; in 2020, especially during the first months of the pandemic period, the incidence of invasive disease saw an historical drop and the change was greatest for children aged 5 to 17 years. Unexpectedly, the preliminary data from the surveillance reports of 2022 showed a monthly increase of the incidence of iGAS infection in children from September to November, thus leading the CDC to issue a health advisory. Different European countries reported to ECDC an increase in iGAS disease in children aged less than 10 years from September 2022, with Ireland, France and UK reporting several deaths, too. Itâs likely that this increase in incidence of iGAS is related to the high circulation of respiratory viruses, especially Respiratory Syncytial Virus (RSV) and seasonal influenza, since the coinfection of viruses can lead more easily to invasive disease.\n\nThe most common clinical presentations of iGAS during the pediatric age include bacteremia, soft tissue infections, Streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis.1 The presence of one other child living in the same house, a varicella zoster virus infection and the use of nonsteroidal anti-inflammatory drugs have been reported in literature as risk factors for iGAS.2,3 Due to the extreme variety of severity of GAS infection, the early diagnosis of invasive disease is often challenging.\n\nThis scoping review aims to analyze the early signs and clinical features of invasive group A streptococcus disease in children, to recognize the prodromal stage of the disease and quickly start the appropriate antibiotic therapy and supportive care.\n\n\nReview questions\n\nThe main review question will be âwhich are the predictors of clinical iGASâ?\n\nThis review will also assess the following sub-questions:\n\n1. What is known about the epidemiology of iGAS?\n\n2. Which are the most frequently reported clinical characteristics of different iGAS?\n\n3. Which outcomes are reported in literature about the different types of iGAS (pneumonia, meningitis, sepsis and abscesses)?\n\n\nInclusion criteria\n\nThis review will include studies performed on children and adolescents (younger than 18 years) with a confirmed diagnosis of iGAS defined as a laboratory isolation of GAS from any normal sterile site or isolation of GAS from a non-sterile site in patients with necrotizing fasciitis or streptococcal toxic shock syndrome.4 We will include children diagnosed with pneumonia, sepsis, abscesses or meningitis, due to GAS invasion.\n\nThe main concept of this review will be the iGAS in all its aspects.\n\nConsidering the severity of the disease, we will not expect to find articles involving patients not hospitalized so we will include only inpatients.\n\nThis review will include both randomized controlled trials and non-randomized controlled trials. All the types of observational studies, prospective and retrospective (including case-control, cohort and cross-sectional studies, small case series or single case reports) will be included.\n\n\nMethods\n\nThe search will be performed by one reviewer. We started our research in January 2023 in the following bibliographic databases: PubMed and SCOPUS. There will be no date restrictions. Only articles written in English will be included. The search strategy will include the following word: âpediatricâ, âiGAS (and its possible clinical manifestations)â and âgroup A Streptococcus Pyogenesâ. Patients younger than 18 years of age will be considered as children or pediatric patients. The search strategy for PubMed is available in the extended data section of this protocol5; the terms used for this search were adapted for use with other bibliographic database.\n\nIf the final analysis were to be performed six months after the bibliographic search, the search string will be launched again to evaluate the presence of new studies to be included in the work.\n\nAfter the search, the studies will be exported to Rayyan. A first screen to exclude duplicates will be performed by one author.\n\nTitles and/or abstracts of studies retrieved using the search strategy will be screened independently by two reviewers to identify studies that could be inserted in the review. Full texts of potentially eligible studies will be retrieved and independently assessed for eligibility by two reviewers. Each researcher will be blinded to the decision of the other researcher. Any disagreement between them over the eligibility of studies will be resolved through discussion and, in case of further disagreement, by discussion with a third reviewer.\n\nAll the studies that will not meet the inclusion criteria will be excluded and a table with the reason why those studies were excluded will be inserted in the final manuscript.\n\nThe results of the search will be reported in the PRISMA flow diagram.\n\nTwo review authors will extract data independently, everyone on a different Excel spreadsheet. Each researcher will be blinded to the decision of the other researcher. When the process will be completed, in case of discordance, any disagreement will be identified and resolved through discussion (with a third author if necessary).\n\nAn Excel file will be used to store data. When available, extracted information will include:\n\n1. study general features: title, author, year of publication, type of study, number of patients included in the study, geographical area where the study has been performed\n\n2. participant general features: sample size of each group, nationality, age, socio-economic status, comorbidities\n\n3. previous clinical manifestations (during the 30 days preceding the diagnosis of iGAS): fever (including days), sore throat, vomiting, rashes, cough and others, known pharyngeal swab positive for Group A streptococcus, and others\n\n4. clinical manifestation of the condition: fever (including days), neurological signs, vomiting, rashes, cough and others\n\n5. a concomitant or previous (during the 30 days preceding the diagnosis of iGAS) viral infections microbiologically confirmed\n\n6. main imaging findings: type of lung involvement at chest X-Ray and/or CT scan, type of CNS involvement at CT scan or MRI, type of skin involvement evaluated by ultrasound or CT scan or MRI, heart (US or CT or MRI)\n\n7. GAS localizations (e.g., lung, central nervous system, blood or skin)\n\n8. characteristics of eventual antimicrobial treatments performed during the 30 days preceding the diagnosis of iGAS (length of therapy, when this has been started and which antibiotic was used)\n\n9. characteristics of eventual antimicrobial treatments performed during the iGAS (length of therapy, when this has been started and which antibiotic was used)\n\n10. adjunctive treatments performed and length of therapy during the iGAS (e.g., steroids or other immunomodulatory medications)\n\n11. outcomes (death, survival; survival with or without sequelae; type of sequelae)\n\nTo report our findings, we will follow Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist.\n\nWe will produce a narrative synthesis of the findings from the studies included in the review describing the results we have obtained and providing our opinion on their interpretation. The selection of the studies for the main narrative synthesis will be performed preferring articles in which the clinical history before the iGAS has been reported; for this part we will include only articles in which the risk factors of iGAS development was evaluated trough multivariable analysis. This selection is motived by the fact that our first interest is the identification of iGAS predictors. If after this selection will be included more than 100 records, original article and those published in the last 5 years will be preferred.\n\nWe will also use tables and charts to summarize both study characteristics and the most important clinical, diagnostics, treatments and outcomes data.\n\nMore specifically, we will summarize our findings using different tables. The first one will include the characteristics of included studies (number of studies, study design, year of publication, characteristics of the study populations, and countries where studies were conducted) and the participant general features. Then we will provide different tables or figures summarizing main data about clinical presentation, imaging characteristics, GAS localization, treatments performed, outcomes and predictors of iGAS.\n\nThis way we hope we will be able to provide a useful document containing what is currently known of pediatric iGAS with the aim of informing clinicians about the general characteristics of these conditions, focusing on risk factors and early clinical features, and guide future research projects to fill current gaps.\n\n\nStudy status\n\nWe launched our research and performed the abstract screening. We are going to start the full text screening.\n\n\nPatient and public involvement\n\nThere was no direct patient and public involvement in this review. However, the key questions that led us implementing this research project were inspired by public discussions started by family associations in the media, highlighting the importance of better comprehension of how iGAS can be recognized earlier in the disease course (before clinical conditions deteriorates and cannot be controlled anymore), or iGAS may also be prevented if this complication is a consequence of a previous unrecognized and untreated GAS infection.\n\n\nStrengths and limitations of this study\n\n\n\n⢠A scoping review can represent the best way to report on the types of evidence that are published in a certain field and our paper will provide an overview of iGAS, focusing on predictors of invasive infection in children.\n\n⢠A scoping review can represent the best way to examine this field to guide future research on this topic.\n\n⢠To report our findings, we will follow the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist to ensure methodological strength to our paper.\n\n⢠Only two databases were screened, and only English paper will be considered limiting the number of papers that will be included.\n\n⢠No critical appraisal neither risk of bias of the included studies will be performed, considering the exploratory role of this paper.",
"appendix": "Data availability\n\nNo data are associated with this article.\n\nFigshare: Supplementary Material.docx, https://doi.org/10.6084/m9.figshare.22094219.v1. 5\n\nThis project contains the following extended data:\n\n- Supplementary Material.docx (Search strategy for PubMed)\n\nData are available under the terms of the Creative Commons Zero âNo rights reservedâ data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nZachariadou L, Stathi A, Tassios PT, et al.: Differences in the epidemiology between paediatric and adult invasive Streptococcus pyogenes infections. Epidemiol. Infect. 2014; 142(3): 512â519. Publisher Full Text\n\nFactor SH, Levine OS, Harrison LH, et al.: Risk factors for pediatric invasive group A streptococcal disease. Emerg. Infect. Dis. 2005; 11(7): 1062â1066. Publisher Full Text\n\nLaupland KB, Davies HD, Low DE, et al.: Invasive group A streptococcal disease in children and association with varicella-zoster virus infection. Ontario Group A Streptococcal Study Group. Pediatrics. 2000; 105(5): E60.\n\nSherwood E, Vergnano S, Kakuchi I, et al.: Invasive group A streptococcal disease in pregnant women and young children: a systematic review and meta-analysis. Lancet Infect. Dis. 2022 Jul; 22(7): 1076â1088. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMariani F: Supplementary Material.docx. figshare. Figure. 2023. Publisher Full Text"
}
|
[
{
"id": "164677",
"date": "16 Mar 2023",
"name": "Damian Roland",
"expertise": [
"Reviewer Expertise Paediatric Emergency Medicine Health Services Research"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important and timely piece of work.\n\nIntroduction:\n\nWhy was the increase in 2022 described as 'unexpectedly\" when an increase was potentially inevitable after a reduction following lockdown?\n\n\"It's likely this increase is related to high circulation of respiratory viruses\" > This is obviously a possibility but why is this more likely than just the fact herd immunity and transmission waned during the pandemic?\nConcept:\n\"The main concept of this review will be the iGAS in all its aspects\" >Â The focus of this review will be the impact iGAS in children and adolescents.\n\nSearch Strategy:\nWhy are only PubMed and SCOPUS being searched? Is there a reason for excluded COCHRANE and CINAHL?\n\nHas a clinical librarian been involved?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
},
{
"id": "279813",
"date": "21 May 2024",
"name": "Navin P Boeddha",
"expertise": [
"Reviewer Expertise Pediatric Infectious Diseases and Immunology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper by Mariani and colleagues is a study protocol to review early signs and clinical features of iGAS in children. This is an important paper because early recognition and early treatment of invasive bacterial infections improve outcome. Apart from early recognition, the review will be much broader, e.g. epidemiology, therapy, and outcome.\nOverall the study protocol is presented clearly. A few minor comments/queries: 1. Introduction, second paragraph: âItâs likely that this increase in incidence of iGAS is related to the high circulation of respiratory viruses, especially Respiratory Syncytial Virus (RSV) and seasonal influenza, since the coinfection of viruses can lead more easily to invasive disease.â Please provide a reference for this statement. I would suggest to weaken this statement as multiple factors may play a role, e.g. pathogenic emm types (Brouwer S, et al., 2023 [Ref1])\n2. Inclusion criteria: why do the authors do not include osteoarticular infections? (Trobisch A, et al., 2022 [Ref 2])\n3. Search strategy: is there a librarian involved?\n4. Data extraction: consider inclusion of the following variables: Immunization status (Varicella vaccination?), previous surgery (Duvekot A, et al., 2019 [Ref 3].), lab predictors  (CRP, PCT, leukocytes, neutrophils).\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/12-209
|
https://f1000research.com/articles/11-1268/v1
|
08 Nov 22
|
{
"type": "Research Article",
"title": "How have Japanese primary care physicians carried out vaccinations against COVID-19? : Attempts at making the non-scalable âscalableâ",
"authors": [
"Shuhei Kimura",
"Sachiko Horiguchi",
"Ryohei Goto",
"Junko Iida",
"Sachiko Ozone",
"Makoto Kaneko",
"Junko Teruyama",
"Yusuke Hama",
"Junji Haruta",
"Junichiro Miyachi",
"Sachiko Horiguchi",
"Ryohei Goto",
"Junko Iida",
"Sachiko Ozone",
"Makoto Kaneko",
"Junko Teruyama",
"Yusuke Hama",
"Junji Haruta",
"Junichiro Miyachi"
],
"abstract": "Vaccine rollouts have been underway to combat the COVID-19 pandemic globally. Based on ongoing interviews with ten primary care physicians âin the fieldâ, this paper elucidates how in practice the vaccinations were carried out in Japan in 2021. We examine what the primary care physicians did to prepare for the rollouts, what problems they faced, and how they responded to these problems. Large-scale vaccination projects are supposed to proceed smoothly and quickly, or to have what Anna Tsing calls âscalabilityâ. In practice, however, they required a variety of tasks for coordination, information sharing, and promotion. Despite feeling stressed by the lack of information and exhausted by the work overload, the primary care physicians carried out the vaccinations as an important service to their patients and communities. The findings of this paper will provide valuable materials for improving future vaccine rollouts.\näžçååœã§æ°åã³ãããŠã€ã«ã¹ææçãžã®å¯ŸçãšããŠã¯ã¯ãã³æ¥çš®ãé²ããããŠãããæ¬çš¿ã¯2021幎ã«ãããŠãæ¥æ¬åœå
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éã«é²ããã¹ãã¯ã¯ãã³æ¥çš®ãããžã§ã¯ãã¯ããã³ã®ãããã¹ã±ãŒã©ããªãã£ãããã€ããšãåæãããŠããããããå®éã«ã¯çŸå Žã§ã®æ§ã
ãªèª¿æŽãæ
å ±å
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å ±äžè¶³ãžã®ã¹ãã¬ã¹ãæ¥åéå€ã«ããç²åŒãæãã€ã€ãå°åããããã€ãæ£è
ãžã®å€§äºãªãµãŒãã¹ãšããŠæ¥çš®ãè¡ã£ããæ¬çš¿ã®ç¥èŠã¯ä»åŸã®ã¯ã¯ãã³æ¥çš®èšç»ãå®æœã®æ¹åã®ããã®è³æãšãªããã®ã§ããã",
"keywords": [
"COVID-19",
"primary care physician",
"Japan",
"vaccine",
"scalability"
],
"content": "I. åºè«ââã¯ã¯ãã³æ¥çš®ãèšé²ãã\n\næ¬çš¿ã¯ãæ°åã³ãããŠã€ã«ã¹ææçïŒä»¥äžãCOVID-19ïŒãžã®å¯ŸçãšããŠéå§ãããã¯ã¯ãã³æ¥çš®ã«ã€ããŠããã©ã€ããªã»ã±ã¢å»ãžã®ç¶ç¶çãªã€ã³ã¿ãã¥ãŒã«ããšã¥ãã(1) æ¥æ¬ã§ã¯ 2021 幎ã«ã©ã®ããã«è¡ããããã©ã€ããªã»ã±ã¢å»ã®ç«å ŽããèŠãŠã©ã®ãããªåé¡ãããããããã¯ããã«ã©ã®ããã«å¯Ÿå¿ããã®ãã®å®æ
ãæŽçããããšã(2) ããã«ãã£ãŠãä»åŸã®ãã³ãããã¯ã«åããŠã®ç¥èŠãæ瀺ããããšãç®çãšããã\n\nCOVID-19 ãåŒãèµ·ãã SARS-CoV-2 ã«å¯ŸããŠã¯ãã¯ã¯ãã³ã®éçºãšæ¥çš®ããã³ãããã¯çµæ¯ã®éµã®ã²ãšã€ãšèãããããã€ãŠãªãã¹ããŒãã§é²ãããããã¯ã¯ãã³ã¯çµæãšããŠæ§ã
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[
{
"id": "155206",
"date": "11 Jan 2023",
"name": "Maho Isono",
"expertise": [
"Reviewer Expertise æå人é¡åŠãå»ç人é¡åŠ"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nãç·æ¬ã æ¬è«æã¯ã³ããçŠã®åæããåœå
ã®ãã©ã€ããªã»ã±ã¢å»ã«å¯ŸããŠç¶ç¶çã«å®æœãããŠããã€ã³ã¿ãã¥ãŒããŒã¿ã«åºã¥ããŠããããŸããã®ç¹ã«ãããŠèè
ãéããèªãã¯å€§å€è²Žéã§ãããšèããŸãããŸãæ¬ç 究ããæå人é¡åŠè
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"responses": [
{
"c_id": "9325",
"date": "24 Feb 2023",
"name": "Shuhei Kimura",
"role": "Author Response",
"response": "ãã®ãã³ã¯å€§å€ãå¿ããäžãæ¬çš¿ã®æ»èªã³ã¡ã³ãããå·çããã ãããŸãæ¬çš¿ã®ã€ã³ã¿ãã¥ãŒããŒã¿ããã³æå人é¡åŠè
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ãªè¡çºã®ç·äœãšããæå³ã§ãããã®ç¹ã第Iç« ã«å çããŸããã"
}
]
}
] | 1
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https://f1000research.com/articles/11-1268
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https://f1000research.com/articles/11-216/v1
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23 Feb 22
|
{
"type": "Research Article",
"title": "The knowledge, attitude and practice level of dental auxiliaries regarding oral health care for pregnant patients in the eastern province of Saudi Arabia",
"authors": [
"Daneah Alibrahim",
"Azza El. Mahalli",
"Daneah Alibrahim"
],
"abstract": "Background: The purpose of this research was to assess the knowledge, attitude, and practice of dental auxiliaries related to oral health care for pregnant patients in the Eastern Province of Saudi Arabia. Methods: A cross-sectional study using a questionnaire survey was conducted. The knowledge, attitude, and practice were rated using the Likert scale out of 5. Knowledge and practice were categorized using Bloomâs cut off point methods (â¥80% is good, and <80% is bad). Concerning attitude, (â¥80% is positive, and <80% is negative). The questionnaires were sent to all dental hygienists and assistants (N=358), and responses were collected from 218. Statistical Package for the Social Sciences (SPSS) software was utilixed to conduct statistical analysis. Results: Out of the 358, 218 responded (response rate = 61%). More than half of the respondents showed relatively good knowledge (57.3%). Most respondents had a positive attitude (89.4%). Regarding practice, approximately two-thirds had a good practice (67.4%). The knowledge score of hygienists was significantly higher than dental assistants, and respondents with experience in treating pregnant patients had significantly higher knowledge scores than others who did not have experience in treating pregnant patients. There is a statistically significant positive correlation between practice scores and education. Conclusions: The findings suggest the need to establish continuous education programs and for dental hygienists and dental assistants to adopt the best practice guidelines on perinatal oral health.",
"keywords": [
"Dental auxiliaries",
"Pregnancy",
"Oral care",
"Knowledge",
"Attitude",
"Practice",
"Saudi Arabia"
],
"content": "Introduction\n\nPregnancy is an experience that most females undergo during their lives, which contributes to physiological and psychological changes. One of the most apparent changes is the one in hormonal levels such as estrogen and progesterone that significantly influences various health issues. Hormonal changes can increase the risk of gingivitis and periodontitis.1â4 Preventing plaque formation during pregnancy is important for both the mother and the fetus.5 Health care practitioners from diverse backgrounds work together during pregnancy to meet the health and well-being of mothers and their developing fetuses.6 Dental professionals such as dentists and dental auxiliaries (dental hygienists and assistants) are well-positioned to provide oral care for pregnant patients and facilitate referrals to other health care providers.3,6 To improve the services provided to pregnant patients, researchers need to understand the knowledge and awareness level of dental auxiliaries regarding oral health care for pregnant patients.6 A few studies have been performed in the United States that discuss dental hygienists' awareness, attitude, and practice towards pregnant patients.6 However, in Saudi Arabia, no studies have been conducted to assess the awareness, attitude, and practice of dental auxiliaries towards pregnant patients. Dental hygienists are well positioned to provide oral care, introduce pregnancy oral health information, and encourage referrals to other health care providers. Thus, the study aims to assess the level of knowledge, attitude, and practice of dental auxiliaries regarding oral health care for pregnant patients in the Eastern Province of Saudi Arabia.\n\n\nMethods\n\nEthical approval was obtained from Imam Abdulrahman Bin Faisal University research ethical review board (IRP-PGS-2020-03-378). Written informed consent was obtained from all subjects who agreed to participate in the study. The confidentiality and privacy of the subjects were maintained.\n\nThis population-based cross-sectional study was conducted at the governmental dental hospitals in the Eastern province of Saudi Arabia from 17 February 2021 to 17 March 2021.\n\nStudy participants were all dental auxiliaries (dental hygienist and dental assistant) at the governmental dental hospitals in the Eastern province of Saudi Arabia.\n\nThe questionnaire was used by Schramm et al. (2016) but modified in the current study.6 It comprised of four sections which included socio-demographic characteristics. Knowledge, attitude, and practice were rated using the Likert scale: 1 = strongly disagree, 2 = disagree, 3 = neutral, 4 = agree, 5 = strongly agree. Knowledge and practice were categorized using Bloomâs cut off point methods (â¥80% is good, and <80% is bad). Concerning attitude, (â¥80% is positive, and <80% is negative).7 The survey was administered via Google forms services to all dental auxiliaries through e-mail. There was no missing data. See the Extended data for a copy of the questionnaire.\n\nA pilot study was conducted on a representative sample of dental auxiliaries from a private hospital. The questionnaire was validated for face and content validity, reliability, and ease of use. Cronbachâs alpha results of the pilot study were acceptable for knowledge (α = 0.778), and attitude (α = 0.791) and good for practice (α = 0.862).\n\nStatistical analysis was performed using SPSS version 23 for Mac. Frequencies and percentages were calculated for categorical variables. The mean and standard deviation were calculated for continuous variables. The median score of 4 was a cut-off point that was considered satisfactory for knowledge, attitude, and practice.8 Mann Whitney and Kruskal Wallis tests were performed to compare the knowledge, attitude, and practice scores. The Spearman rho test was used to correlate the knowledge, attitude, and practice scores with age, education, and experience. A p-value of â€0.05 was considered statistically significant.\n\n\nResults\n\nOf the total of 358, 218 responded, resulting in a response rate of 61% which is considered excellent in most circumstances.9\n\nTable 1 shows the socio-demographic characteristics of study participants.\n\nTable 2 shows that more than half of the study participants (57.3%) had good knowledge regarding oral health care for pregnant patients. Concerning the attitude of dental practitioners towards the pregnant patients, (89.4%) had a positive attitude. Finally, approximately two-thirds of participants had good practice (67.4%).\n\nTable 3 shows that the knowledge score of hygienists was higher than dental assistants and of those experienced in treating pregnant women was higher than participants without experience and these differences were significant (P < 0.05).\n\n* P †0.05.\n\nTable 4 shows that there is no statistically significant difference in the attitude score by gender, nationality, marital status, specialty, workplace nor experience in treating pregnant females (P > 0.05).\n\nTable 5 shows that there is no statistically significant difference in the practice score by gender, nationality, marital status, specialty, nor workplace (P > 0.05). However, there is a significant difference between participants with experience in treating pregnant patients and those without experience (P < 0.05).\n\n* Statistically significant at P †0.05.\n\nTable 6 shows the results of the correlation between knowledge and age, education and experience. There is a positive correlation between the knowledge score and age, education, and experience; however, these correlations are not statistically significant.\n\nTable 7 shows the results of the correlation between attitude and age, education and experience. There is a negative correlation between the attitude score and age, education, and experience; however, these correlations are not statistically significant.\n\nTable 8 shows the results of the correlation between practice and (age, education and experience). There is positive correlation between the practice score and age, education and experience. However, the only statistically significant correlation is with education (P < 0.05).\n\n* P †0.05.\n\n\nDiscussion\n\nPregnant women should receive routine and emergency dental treatment; however, there is limited information about the current practices among dental auxiliaries providing care to pregnant women.10\n\nIn this study, dental auxiliaries demonstrated relatively good knowledge (57.3%) regarding oral care provided throughout pregnancy. In addition, they agreed that women should receive preventive dental care during pregnancy (75.2%), which is consistent with the available literature and studies on the subject.11 Of the dental auxiliaries in this study, only 69.7% advocated for the risk of radiographs during this period. Similar disagreement about the use of radiographs for pregnant women has been identified in other studies.11,12 The current findings demonstrated that the knowledge score of hygienists was significantly higher than dental assistants (p = 0.006). This finding is consistent with a study conducted in North Carolina in 2008 where respondents with low or moderate levels of knowledge were significantly less likely to provide comprehensive treatment for pregnant women than those with high levels of knowledge.13\n\nDental auxiliaries had a very positive attitude toward perinatal oral health (89.4%). Other studies also found almost universal agreement that dental treatment should be included in prenatal care, with most dentists and dental auxiliaries having favorable attitudes toward pregnancy-specific counselling.6,11-13 The present findings revealed that 81.6% of participants believe that they need educational material for pregnant patients. These findings were consistent with the literature where dental auxiliaries required continuing education to improve their awareness about pregnant womenâs oral health.6,11,14\n\nThe results of this study revealed that only 36.3% of study participants practiced administration of anesthetic injections. This contradicted previous study results where benzocaine, procaine and lidocaine were administered safely.12,15 In addition, the current study showed that there was a statistically significant correlation between practice and level of education. This finding has been supported by another study which agreed that continuous dental education could be a useful strategy in improving dental practice.12\n\nIn this study, only 218 dental auxiliaries participated and thus the results may not be representative of the dental auxiliaries in the kingdom, and the findings may be not generalizable.\n\n\nConclusion\n\nDental auxiliaries had positive knowledge, attitudes, and practices regarding offering oral health care to pregnant women. The research results confirmed that many dental auxiliaries shared the desire for continuing education on oral health care during pregnancy to improve oral health practices. Preventive measures for pregnant women would be beneficial not only for the mothers but also for their babies.\n\n\nData availability\n\nFigshare: main study 2.sav, https://doi.org/10.6084/m9.figshare.17197283.v2.16\n\nThis project contains the following underlying data:\n\n- Main study 2.sav (Data for gender, age, nationality, marital status, education, specialty, work place, experience in treating pregnant patients, and questions 11 to 36)\n\nFigshare: main study 2.sav, https://doi.org/10.6084/m9.figshare.17197283.v2.16\n\nThis project contains the following extended data:\n\n- Survey for magazine.docx (Questionnaire)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nTurton M, Africa CW: Further evidence for periodontal disease as a risk indicator for adverse pregnancy outcomes. Int. Dent. J. 2017; 67(3): 148â156. PubMed Abstract | Publisher Full Text\n\nNIH: Periodontal (gum) disease: Causes, symptoms, and treatments. 2017. Reference Source\n\nBrown A: Access to oral health care during the perinatal period: A policy brief. 2008. Reference Source\n\nJared H, Boggess KA: Periodontal diseases and adverse pregnancy outcomes: a review of the evidence and implications for clinical practice. American Dental Hygienists' Association. 2008; 82(3): 1â20.\n\nBakhshi M, Tofangchiha M, Bakhtiari S, et al.: Oral and dental care during pregnancy: A survey of knowledge and practice in 380 Iranian gynaecologists. J. Int. Oral Health. 2019; 11(1): 21â27. Publisher Full Text\n\nSchramm SA, Jacks ME, Prihoda TJ, et al.: Oral care for pregnant patients: A survey of dental hygienists' knowledge, attitudes and practice. J. Dent. Hyg. 2016; 90(2): 121â127. PubMed Abstract\n\nBloom BS: Taxonomy of educational objectives. Vol. 1: Cognitive domain. New York: McKay; 1956; 20(24): 1.\n\nBarua A: Methods for decision-making in survey questionnaires based on Likert scale. Journal of Asian Scientific Research. 2013; 3(1): 35â38.\n\nKiess HO, Bloomquist DW: Psychological research methods: A conceptual approach. Allyn & Bacon; 1985.\n\nKumar J, Samelson R: Oral health care during pregnancy recommendations for oral health professionals. N. Y. State Dent. J. 2009; 75(6): 29â33. PubMed Abstract\n\nGeorge A, Ajwani S, Bhole S, et al.: Knowledge, attitude and practises of dentists towards oral health care during pregnancy: A cross sectional survey in New South Wales, Australia. Aust. Dent. J. 2017; 62(3): 301â310. PubMed Abstract | Publisher Full Text\n\nHuebner CE, Milgrom P, Conrad D, et al.: Providing dental care to pregnant patients: A survey of Oregon general dentists. J. Am. Dent. Assoc. 2009; 140(2): 211â222. Publisher Full Text\n\nDa Costa EP, Lee JY, Rozier RG, et al.: Dental care for pregnant women. J. Am. Dent. Assoc. 2010; 141(8): 986â994. Publisher Full Text\n\nUmoh AO, Azodo CC: Nigerian dentists and oral healthcare of pregnant women: Knowledge, attitude and belief. Sahel Medical Journal. 2013; 16(3): 111â115. Publisher Full Text\n\nTurner MD, Singh F, Glickman RS: Dental management of the gravid patient. N. Y. State Dent. J. 2006; 72(6): 22â27. PubMed Abstract\n\nAlibrahim D: main study 2.sav. figshare. Dataset. 2021. Publisher Full Text"
}
|
[
{
"id": "124732",
"date": "25 Feb 2022",
"name": "Mohammad Abdul Baseer",
"expertise": [
"Reviewer Expertise Dental Public Health",
"Preventive Dentistry",
"Clinical Trials."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have made a good effort in conducting research on the topic that is not fully addressed in KSA, however:\nAuthors should improve the article's English and grammar. Spelling mistakes in the abstract and other areas should be corrected. The introduction should mention the intended benefits of the study. Under the materials and methods section sample size calculations and sampling techniques utilized in the study should be elaborated. How did the authors obtained email addresses of the participants should me mentioned in the study. Descriptive analysis of each item in the questionnaire should be presented. Normality assessment of the data should be indicated before deciding to opt for non-parametric analysis. Results should also include correlation test among knowledge, attitude and practices. The discussion is very brief, authors should elaborate it by including articles published from Saudi Arabia and elsewhere. Authors should include more study limitations. References should be updated with more than half being published within last 2-3 years.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "7998",
"date": "28 Mar 2022",
"name": "Daneah Alibrahim",
"role": "Author Response",
"response": "Dear Dr. Mohammad Abdul Baseer Thank you for the comments, I really appreciate it. I have addressed your comments and submitted the new version. best regards"
}
]
}
] | 1
|
https://f1000research.com/articles/11-216
|
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