Datasets:

VCY019
/

Numeracy-Probing

id stringlengths 3 9	source stringclasses 1 value	version stringclasses 1 value	text stringlengths 1.54k 298k	added stringdate 1993-11-25 05:05:38 2024-09-20 15:30:25	created stringdate 1-01-01 00:00:00 2024-07-31 00:00:00	metadata dict
258324270	pes2o/s2orc	v3-fos-license	Schistosomiasis in Europe The purpose of this review is to provide an overview of the burden of schistosomiasis in the European continent. It discusses three subjects: the endemic forms of non-human schistosomiasis in Europe; the introduction of transmission of human schistosomiasis into Europe; and the occurrence of imported cases of human schistosomiasis. Europe is not endemic for human schistosomiasis; nevertheless, it is affected by the disease in multiple ways, although the magnitude of the burden remains elusive because of gaps in surveillance and reporting. Schistosomiasis is a global neglected disease prevalent in tropical and subtropical areas. As of 2022, it is estimated that 251 million people require preventive chemotherapy for schistosomiasis, 90% of whom live in Africa. In Europe, human schistosomiasis is frequently detected in migrants from endemic countries who reach the continent. Additionally, outbreaks due to local transmission can sporadically occur following the introduction of schistosomes in one of the many freshwater bodies in southern Europe where competent snail hosts are found. Finally, human cercarial dermatitis is frequently occurring in Europe, because of the presence of avian schistosomiasis in several countries across the continent. A stronger epidemiological surveillance and reporting system, coupled with more surveys on humans and snails, can contribute to better assess and characterize the burden of schistosomiasis in Europe. Introduction Schistosomiasis is a neglected tropical disease caused by blood flukes (trematode parasites) belonging to the genus Schistosoma. It is a vector-borne disease transmitted by several species of freshwater snails. Human infection is associated with two main clinical forms: intestinal schistosomiasis and urinary (or urogenital) schistosomiasis [1•]. The species responsible for intestinal schistosomiasis in humans are six: S. mansoni, transmitted by Biomphalaria spp. snails; S. japonicum, transmitted by Oncomelania spp. snails; S. mekongi, transmitted by Neotricula spp. snails; S. malayensis transmitted by Robertsiella spp.; and S. guineensis and the related S. intercalatum, transmitted by Bulinus spp. The only species responsible for urinary (or urogenital) schistosomiasis in humans is S. haematobium, transmitted by Bulinus spp. snails [1•]. More than 10 other schistosome species are parasites of animals, in which they cause forms of schistosomiasis that are endemic in several countries and may occasionally infect humans. Nevertheless, parasitic larvae rarely reach adult stage in the human host and are only responsible for mild and transient disease. Schistosomiasis is a global disease mainly prevalent in tropical and subtropical areas. According to WHO, at global level, over 251 million people require preventive chemotherapy for schistosomiasis, with approximatively 90% of them living in Africa. Globally, 78 countries are considered endemic for schistosomiasis by WHO [2•]. Nevertheless, the actual number of endemic countries could be higher. For example, cases of schistosomiasis have been reported recently in Nepal [3,4], in Myanmar [5], as well as in Europe (please see further below). This review focuses on schistosomiasis in Europe. It discusses three subjects: (2) The introduction of transmission of human schistosomiasis into Europe (3) The occurrence of imported cases of human schistosomiasis Avian Schistosomiasis and Other Forms of Non-human Schistosomiasis Although human schistosomiasis is not endemic in Europe, avian schistosomiasis is present in several European countries, including Czech Republic, Denmark, France, Germany, Iceland, Poland, Spain, and Switzerland [6, 7•]. Its distribution is in fact global and outside Europe, it has been documented in several countries including Argentina, Australia, Canada, Chile, Iran, Japan, the Netherlands, New Zealand, Saudi Arabia, South Africa, and the USA [6,[8][9][10]. Trichobilharzia spp., Ornithobilharzia spp., and Bilharziella spp. are the most common genera responsible for avian schistosomiasis in Europe, while the most common molluscan intermediate hosts belong to the pulmonate snail families Physidae (e.g., Physa spp.), Lymnaeidae (e.g., Radix spp.), and Planorbidae (e.g., Biomphalaria spp.) [6]. As the word "avian" indicates, the natural hosts are represented by several aquatic birds, such as ducks of the genera Anas, Aythya, Cairina, and Spatula; swans of the genus Cygnus; and geese of the genus Anser [11,12]. Avian schistosomes may be responsible for disease in their natural animal hosts; for example, the pathogenic effects of Trichobilharzia regenti on the central nervous system of Anas, Cairina, and Spatula are well-known and studied [13]. Adult schistosomes reside in the mesenteric veins of their animal host. Eggs reach the intestine, are passed in feces, and released in the environment. In contact with water, eggs hatch and liberate miracidia which in turn penetrate into a suitable snail intermediate host and develop into cercariae. Finally cercariae are released in water where they can infect animal hosts and occasionally human hosts (Fig. 1). Surface freshwaters are typically those where cercariae are found. Besides avian schistosomes, a few genera of the family Schistosomatidae having mammals as natural hosts are known to be responsible for cercarial dermatitis. Among them, Orientobilharzia spp. has been found in Europe (Hungary, Russia), where Radix auricularia has been identified as the intermediate host; natural final hosts are ungulates such as cervids (e.g., deers) [14][15][16]. The distribution of mammal schistosomiasis in Europe and its relevance to human health are certainly less important than in the case of avian schistosomiasis. Nevertheless, it should be noted that S. bovis, a parasite causing intestinal schistosomiasis in ruminants, is present in several areas of Mediterranean Europe, where its intermediate hosts are Bulinus spp. and Planorbarius metidjensis [17]. Although S. bovis is not associated with infection in humans, it can hybridize with other Schistosoma species and produce infective offspring. Notably, S. haematobium/S. bovis hybrids have been found to be responsible for human infections in Corsica [18•] (see below). Epidemiology Humans can be accidental hosts of avian schistosomes, as their larval stages (cercariae) can penetrate human skin. However, when in the human body, avian cercariae die in the skin, being entrapped by the host's immune response; as such, they do not mature into adults and are not able to complete the life cycle within the human accidental host. Nevertheless, cercariae are responsible for a cutaneous, localized inflammatory response associated with their penetration of the human skin. Human cercarial dermatitis, commonly referred to as swimmer's itch, is frequently reported from several European countries; it is often underreported because of its mild health impact and as such likely to occur in other countries in addition to those mentioned above. The earliest known reports of human cercarial dermatitis ("koganbyo" or "lakeside disease") can be traced in the late nineteenth-century Japan, a country then endemic for human schistosomiasis due to S. japonicum [19]. In 1928, William W. Cort first described the occurrence of human cercarial dermatitis due to non-human schistosomes at Douglas Lake, Michigan, USA [19], while the first documented outbreak occurred at Clear Lake, Manitoba, Canada, in 1934, with over 50,000 people affected [19]. In Europe, the earliest reports of human cercarial dermatitis date back to the 1930s, when Brumpt described a case of "dermatite des nageurs" due to Cercaria ocellata in France [20]. Case reporting continued from other European countries over the following decades; in Denmark, for example, the first case of human cercarial dermatitis was reported in the 1950s, although avian schistosomiasis had already been documented in the 1930s [7 •]. While human cercarial dermatitis due to mammal schistosomes has not been reported from Europe, it cannot be excluded that human infections by cercariae of mammalian schistosomes can lead to similar pathologies as those caused by avian schistosome. In fact, human cercarial dermatitis from Orientobilharzia spp. has been reported from non-European countries such as Iran [21]. The burden of human cercarial dermatitis in Europe is not well known. The mildness of the disease entails its frequent under-reporting. Nevertheless, the condition is known to be widespread across the continent. It can be considered an occupational disease in people whose profession entails regular contact with water, as in the case of fishermen and people working on the docks [16]. Human cercarial dermatitis in Europe typically occurs during the summer (June to September), when bathing for recreational purposes is common as a mean to seek refreshment from warm temperatures. Signs and Symptoms Infection with avian schistosomes follows contact with infested water (lakes, ponds, or reservoirs; occasionally shallow marine waters too), usually for occupational or recreational purposes. Symptoms of human cercarial dermatitis include reddening and itching of skin surfaces exposed to water; the most common locations are legs and feet. This is an indication of initial penetration of the cercariae. The onset of symptoms usually occurs between a few minutes (when the person is still in the water or immediately after emerging) and a maximum of 24 h after exposure. Lesions are initially erythematous maculae (1-2 mm in diameter) due to local inflammation, but rapidly evolve into larger papulae (3-5 mm in diameter) and after a few additional hours may become vesicular (1-2 mm in diameter). Itching may be important and be associated with insomnia [16]. Signs and symptoms are usually self-limiting; they peak within 1-3 days after exposure, begin to resolve after 5-7 days, and may last 1-3 weeks. The resolution phase may be characterized by a reddish spot which gradually disappears; post-inflammatory hyperpigmentation (hypermelanosis) may persist for months or indefinitely [16]. The development of lesions is mediated by the human host's immune responses, indicating the occurrence of hypersensitization and the development of allergic-type, immunogenic reactions [7•]. The first exposure may not be associated with signs and symptoms but causes a sensitization, which triggers a more rapid and severe onset of signs and symptoms in people with a history of previous contact with cercariae, especially in the case of repeated exposure. Sensitization may persist for year even in absence of new exposures [16]. Infections with a large number of cercariae may also cause fever, limb swelling, nausea, and diarrhoea [16]. Scratching the affected areas may result in secondary bacterial infections, especially in the case of ruptured vesicles. Recent studies in animal models have demonstrated that cercariae may occasionally survive in non-compatible hosts for days and weeks and reach other organs (including the central nervous system, heart, kidney, intestine, liver, and lungs) where they cause damage to host tissues. Such ectopic locations are more frequent in primary infections, as milder inflammatory reactions allow cercariae to escape from the skin and migrate further. Further investigations are required to assess the relevance of these findings to infection in humans [16]. Diagnosis and Treatment The timeline, appearance, and distribution of the lesions body, as well as the history of exposure to a freshwater body should suggest the diagnosis of human cercarial dermatitis and enable a differentiation from similar skin lesions resulting from insect bites or contact dermatitis [16]. Most lesions resolve spontaneously and do not require any treatment; however, good hygiene to prevent itching and secondary infections is important. Topical antipruritic agents, antihistamines, or corticosteroids can be used to relieve symptoms. Systemic antihistamines or corticosteroids may be required in case of severe clinical pictures [22•]. Praziquantel is the treatment of choice for all forms of human schistosomiasis [23 •]. However, it is not effective against the larval stages of Schistosoma spp. [24]; as such, its administration to patients suffering from human cercarial dermatitis is not recommended. Introduction of Transmission of Human Schistosomiasis into Europe Human schistosomiasis is not endemic in Europe. Nevertheless, foci of transmission have been occasionally documented as a result of the introduction of parasites into freshwater bodies populated by competent snail hosts. In the course of the twentieth and twenty-first century, transmission of S. haematobium has been reported from three countries: Portugal, Spain, and France. In Portugal, transmission was limited to one locality, Estoi, located in the southern Algarve province. Cases were first detected in the 1920s, and transmission was still reported in 1948. Nevertheless, it has been considered extinct since a survey was carried out in 1966 [25], while the last patient was cured in 1967 [26]. It is unclear how recent the establishment of transmission in Portugal could be considered; most probably, the parasite had been introduced by travelers from Angola (then a Portuguese colony), and autochthonous transmission could occur because of the presence of local strains of aquatic snails [ [31] has been developed in order to better delineate the potential areas of transmission. The third documented outbreak due to autochthonous transmission of schistosomiasis in Europe occurred in Corsica, France, in 2011 [29,30]. Local transmission was confirmed in 2013, and more than 100 infected local cases and tourists from several European countries who had bathed in the natural pools along the Cavu river, near Sainte-Lucie-de-Porto-Vecchio, were identified [32]. Further investigations showed that a hybrid of S. haematobium and S. bovis is responsible for the transmission, while the intermediate host is B. truncatus. Although S. bovis is naturally present in Mediterranean Europe, it is considered more likely that hybridization may actually have taken place not in Corsica but elsewhere, likely in Senegal, from where hybrid parasites would have been introduced [18 •, 33,34]. Transmission is apparently still ongoing in Corsica, and a new focus different from the Cavu river [35, 36•], the Solenzara river, also located in the south-eastern part of the island, has been identified. The fact that a hybrid parasite is involved might explain why its transmission cycle remained active on the island; it may infect local livestock, which would act as reservoirs of the disease [34]. The risk of further introduction of transmission of urogenital schistosomiasis in Europe is significant in reason of the intensification of human travels (e.g., migration) and the widespread presence of Planorbidae strains susceptible to S. haematobium infection in several Mediterranean countries. The chance that infected people come into contact with freshwater bodies populated by susceptible snails is likely to increase in the future. Notably, presence of Bulinus spp. has been demonstrated in Cyprus, France, Greece, Italy, Spain, as well as Portugal [26,31,37,38], all countries subject to flows of migrants from endemic countries. In addition, the area of distribution of snails can be further expanded by climate change [39,40], which can favor adaptation of snails to new environmental niches, as well as by transportation of snails via aquatic plant trade, or migrant birds. Introduction of transmission of other species such as S. mansoni in Europe has not been documented so far. Nevertheless, Biomphalaria tenagophila, which is involved in transmission of S. mansoni in Brazil, has been identified in Romania [41]. In addition, some of the snails susceptible to this infection such as B. glabrata, B. traminea or B. tenagophila, as well as B. pfeifferi, an intermediate host of S. mansoni in South America, have proven their capacity to invade new continents [42]. In order to prevent the establishment of transmission of Schistosoma spp. in Europe, the following public health measures can be considered: -Regular malacological surveys including use of environmental DNA techniques in areas where competent snails can be found [43]. -Reinforcement of the epidemiological surveillance: screening of migrants reaching Europe and screening of tourists back from endemic countries [44]. Importation of Cases of Human Schistosomiasis into Europe Every year, large numbers of people travel to European countries for different reasons and purposes. Without considering shorts stays for tourism, and limiting our analysis to European Union countries, between 2 and 3 million people settle in Europe every year. Approximately 90-95% are legal immigrants moving to Europe for work (45%), family (24%), education (12%) asylum (9%), or other reasons (10%). The remaining 5-10% reach Europe through irregular border crossings, including land border crossings and sea crossings [45]. Among those who are issued a first residence permit (legal immigrants), the most frequent countries of origin are Ukraine, Morocco, Belarus, India, Russia, Brazil, Turkey, China, Syria, and USA. Among such countries, only Brazil and China are still reporting autochthonous cases of schistosomiasis [45, 46•]. Among those reaching Europe through irregular border crossings, over 50% come from five countries that are either non-endemic or no-longer endemic for schistosomiasis: Syria (23.2%), Afghanistan (8.4%), Tunisia (8.3%), Morocco (8.2%), and Algeria (6.9%); the first endemic countries represented is Egypt, which however accounts for only 4.6% of the arrivals. Several African endemic countries follow with smaller proportions [45]. In general, it is therefore possible to conclude that the number of immigrants to Europe proceeding from countries endemic for schistosomiasis is quite small; consequently, the number of imported cases of schistosomiasis remains overall modest if compared to the global burden of disease attributable to schistosomiasis. We should also highlight that, irrespective of the numbers, a person with schistosomiasis living in Europe is unlikely to represent a public health problem for anyone, considering that it is almost impossible for Schistosoma spp. to find suitable environmental conditions in European countries to complete the transmission cycle and infect someone else, although exceptions exist (please see above). Nevertheless, the proportion of immigrants from endemic countries that are diagnosed with schistosomiasis is not small, reflecting the high level of transmission as well as the limited access to diagnosis and treatment in countries of origin. Most of the information on imported schistosomiasis comes from surveys published in scientific literature, with the limitations that this non-systematic method for collecting information often entails. A study found that among African immigrants newly arrived to Spain from October 2004 to February 2017, the proportion of those infected with schistosomiasis was 12.3% [47]. Other published studies estimated the prevalence of infection among immigrants from sub-Saharan Africa to Italy to 17% [48], to 10% among immigrants from Mali and Senegal, and to 1% among the general immigrant population, when assessed by parasitology [49]. Another study found a seroprevalence of 10.2%, although only S. mansoni antigens were used [50]. Among 462 recently arrived asylum seekers screened in Italy between 2014 and 2015, 21.2% was positive to at least one test for schistosomiasis [51]. If we focus on studies carried out among people experiencing signs and symptoms of diseases, another study carried out in Italy found that among immigrants seeking care in nine infectious and tropical diseases sentinel centers across the country over a period of 7 years (2011-2017), 47.2% (1350/2858) was diagnosed with schistosomiasis [52]. Those infected are also usually found to suffer from advanced morbidity, indicating that management is often inappropriate in countries of origin, or screening implemented late upon arrival in Europe. For example, a survey [53] found that 47.6% of a group of immigrants to Italy aged 18-29 years infected with S. haematobium had bladder masses at ultrasound examination. Again from Italy, another survey [54] found that a high proportion of patients diagnosed with schistosomiasis, both asymptomatic and symptomatic, were screened or tested only several months after arrival in Italy and most of them presented with clinical apparent disease. A third survey [50] found that 68% of migrants to Italy suffering from urogenital schistosomiasis had urinary tract abnormalities when screened by ultrasonography. In France, Leblanc et al. (2017) [55] reported that 37/40 (93%) immigrant children with a diagnosis of 1 3 schistosomiasis had a chronic urinary form with hematuria. In Spain, Roure et al. (2017) [56] concluded that morbidity associated with chronic long-term schistosomiasis is frequent among African immigrants in non-endemic countries. Some investigations were carried out among specific population groups, such as children or women. In Paris, France, a survey conducted among both symptomatic and asymptomatic recently immigrated children estimated the prevalence of schistosomiasis at 24.3%, by using multiple tests [57]. A few studies have also focused on gender issues. For example, Roure et al. (2022) [58•] report on an assessment conducted among a migrant population coming from schistosomiasis-endemic countries and settled in metropolitan Barcelona, Spain. Serology for schistosomiasis among the whole sample was positive in 222/405 (54.8%). That proportion was slightly higher (58.8%) among the 51 women in that population (30/51). Positive women also showed a higher prevalence of gynecological signs and symptoms compared to the seronegative women (96.4% vs. 66.6%). In addition to the more common infections with S. mansoni and S. haematobium, other rarer forms of imported schistosomiasis, such as the one caused by S. japonicum, have also been reported from Europe, as a recent review carried out in Italy has shown [59]. Few studies investigated the relative proportion of cases of schistosomiasis detected among non-European immigrants compared to the total number of cases detected in Europe. The European Network for Tropical Medicine and Travel Health conducted a sentinel surveillance study on 1465 cases of imported schistosomiasis between 1997 and 2010 [60]. The results show that 486 (33%) cases were identified among European travelers, 231 (16%) among long-term expatriates, and 748 (51%) among non-European immigrants. A survey also tried to assess awareness of schistosomiasis among health professionals; an investigation including specific questions about the diagnosis and management of "tropical" urological diseases was carried among European urologists, showing limited knowledge of schistosomiasis and its associated morbidity [61]. In conclusion, it can be said that while the number of cases of schistosomiasis imported into Europe is overall relatively small, its proportion among people originating from endemic countries is quite high. Information available remains limited, however, for the reason that schistosomiasis is not a disease under surveillance in European Union or European Economic Area (EU/EEA) countries, and the relevant information, when available at country level, is not shared with institutions such as the European Centre for Disease Prevention and Control (ECDC) or WHO for compilation. Most data available on schistosomiasis in Europe are consequently generated by surveys. Nevertheless, these are not likely to generate an accurate picture of prevalence and incidence of new cases. For example, they are implemented among selected population groups such as people experiencing symptoms of schistosomiasis or poor health in general, or those attending health facilities for any reason; in addition, they often employ different diagnostic techniques that may hamper a proper comparison and interpretation of information. Finally, a real appreciation of the burden of imported schistosomiasis in Europe is affected by the fact that most studies on this subject are available from a small number of European countries only, mainly those through which irregular immigrants from schistosomiasis-endemic countries (mainly African countries) enter the continent, that is, Italy and Spain. It should also be noted that in general, information on imported schistosomiasis in European countries not belonging to the EU/EEA is very limited. In terms of normative directions, it should be noted that guidance published by the European Union advises that "serological screening and treatment (for those found to be positive) [should be offered] to all migrants from countries of high endemicity in sub-Saharan Africa, and focal areas of transmission in Asia, South America and North Africa," on the grounds that "it [is] likely to be effective and cost-effective to screen child, adolescent and adult migrants" [44,62]. Nevertheless, information on the actual implementation of this recommendation is not available. One of the reasons could be that there are no standard EU guidelines or standard operating procedures for the screening and treatment of schistosomiasis and consequently few examples of practice [44]. Ireland is reportedly the only EU/EEA country with a published general guidance for screening and treatment of schistosomiasis in asymptomatic migrants. The United Kingdom is the only other European country to have such guidance [44]. Conclusions The burden of schistosomiasis in Europe is likely to be larger than currently reported or estimated [63]. Reasons include the underreporting of human cercarial dermatitis because of its mild associated morbidity, the scarcity and heterogeneity of data on schistosomiasis among people originating from schistosomiasis-endemic countries, and the possibility that small outbreaks of autochthonous transmission go unnoticed as probably associated with low intensity of infection and therefore mild symptoms. A stronger involvement of health services in terms of epidemiological surveillance and reporting system, coupled with more surveys on humans and snails, can contribute to addressing the elusive burden of schistosomiasis in Europe. Conflict of Interest The authors declare no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by either author. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.	2023-04-26T15:16:05.736Z	2023-04-24T00:00:00.000	{ "year": 2023, "sha1": "8de28cea19431ba66c7cacd5e81946e0747518f3", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s40475-023-00286-9.pdf", "oa_status": "HYBRID", "pdf_src": "Springer", "pdf_hash": "3cea6f058fc208f349454278afc35fa7bc3c6562", "s2fieldsofstudy": [ "Medicine", "Environmental Science" ], "extfieldsofstudy": [] }
239546005	pes2o/s2orc	v3-fos-license	The Effect of Volume Controlled and Pressure Controlled Ventilation Modes on Cerebral Oximetry and Blood Gas Status in Laparoscopic Cholecystectomy, A Randomized Controlled Trial Emre Badur Anesthesiology and Reanimation Department, Sisli Hamidiye Etfal Training and Researche Hospital, University of Health Sciences, Istanbul, Turkey Mustafa Altınay (  m_altinay@yahoo.com ) Anesthesiology and Reanimation Department, Sisli Hamidiye Etfal Training and Researche Hospital, University of Health Sciences, Istanbul, Turkey Pınar Sayın Anesthesiology and Reanimation Department, Sisli Hamidiye Etfal Training and Researche Hospital, University of Health Sciences, Istanbul, Turkey Ayşe Surhan Çınar Anesthesiology and Reanimation Department, Sisli Hamidiye Etfal Training and Researche Hospital, University of Health Sciences, Istanbul, Turkey leyla türkoğlu Anesthesiology and Reanimation Department, Sisli Hamidiye Etfal Training and Researche Hospital, University of Health Sciences, Istanbul, Turkey tuğba yücel Anesthesiology and Reanimation Department, Dr.Sadi KONUK Training and Researche Hospital, University of Health Sciences, Istanbul, Turkey Background Since the laparoscopic methods have been introduced to the surgical operations, laparoscopic cholecystectomy has become the golden standard in cholelithiasis surgery. 1 For laparoscopic surgery, carbon dioxide (CO 2 ) insu ation is used which increase the intra-abdominal pressure. The arterial oxygenation, the functional residual capacity and the lung compliance will be affected and may be resulted with cardiovascular events. 2,3 Volume-controlled ventilation (VCV) and pressure-controlled ventilation (PCV) are two mechanical ventilation modes that can be used along with their own advantages and disadvantages. 3 The VCV needed a pre-determined tidal volume (TV). The risk of lung damage is the main concern. In contrast the PCV avoid from excess respiratory tract pressure which applied to the lung. However TV may become unstable. Both techniques previously evaluated if one provide lower respiratory work and better tissue oxygenation. Some studies indicated that PCV is better for arterial and tissue oxygenation. 4 Along with the arterial gas results, near infrared spectroscopy (NIRS) is used to evaluate the depth of anesthesia by measuring the oxygenation change at the tissue level in the prefrontal cortex. 5 Although NIRS was used in different surgeries, their use in laparoscopic abdominal surgery is extremely limited. 5,6 In the available literature we could not be able to nd a study which evaluates the effectiveness of perioperative ventilation modes with the NIRS method. The aim of present study was to compare VCV and PCV modes with NIRS cerebral oximetry and arterial gas results in laparoscopic cholecystectomy A prospective, randomized study was conducted in the Sisli Hamidiye Etfal Training and Research Hospital between March and July 2020. Study was started after obtaining approval from the local ethics committee with the approval number: 1496. Registration of study at ClinicalTrials.gov was made at 25/01/2021 with the NCT04723043 number. Informed consent was taken from all patients. All procedures that performed in our study were made in accordance with the ethical standards of the Helsinki declaration (2008). Sample size calculation and randomization Considering the difference in large effect size (effect size=0.8) between the groups, the sample size was calculated as 70 cases in total for 95% Power, alpha signi cance level 0.05. Randomization was done with closed envelopes before the procedure. Inclusion and exclusion criteria For the study period patients that underwent elective laparoscopic cholecystectomy were enrolled for the study. Patients aged between 18 and 65, with American Society of Anesthesiology (ASA) score 1 and 2, body mass index (BMI) <30 kg / m2, were included. Patients who did not give informed consent, patients who underwent previous thoracic/abdominal surgery, patients who underwent emergency laparoscopic cholecystectomy, patients who have ASA score ≥3, hematocrit value ≤ 30 and, BMI> 30 kg / m2 were excluded. Patients with a history of cardiac, neuromuscular, hepato-renal, endocrine, major pulmonary disease (de ned as a decrease in capacity or ow rates below 70% in pulmonary function tests) were also excluded. Patients who returned to laparotomy for surgical reasons after starting laparoscopically, who developed perioperative hemodynamic instability and who used respiratory mechanics outside the study protocol were excluded from the study. Primary-Secondary Outcomes The primary outcomes of the study were the cerebral oxygenation measured with NIRS, peak pressure and plateau pressure of the patients in both groups, the secondary ndings were the patients' SpO 2 , endtidal carbon dioxide and partial oxygen pressure in arterial blood gases. Preoperative care All patients underwent standard anesthesia evaluation for the procedure. Premedication was done with 0,07 mg/kg intravenous midazolam. Intraoperative care Single derivation electrocardiogram, pulse-oximetry, noninvasive arterial pressure and EtCO 2 parameters were monitored. NIRS monitoring was performed using a Masimo (Irvine, CA, USA) device. NIRS cerebral probes were placed in the right and left frontal regions. A 20-gauge cannula was inserted into the radial artery. Anesthesia induction is by intravenous administration of 2 mg/kg propofol, 1 mg/kg lidocaine, 1.5 mcg/kg fentanyl and 0.6 mg/kg rocuronium bromide. Anesthesia maintenance was done with sevo urane %2 and remifentanil 0.15-0.25 mcg/kg/hour. During the maintenance process, the oxygen-air ow was set to 4 lit/min and the FiO 2 set to 40%. During anesthesia, mechanical ventilation was applied to the patients with a Drager (Medical, Lübeck, Germany) brand device. Mechanical ventilation settings applied to all patients were adjusted according to ideal body weight. In the P group, inspiratory pressure (P insp ) was set to create a tidal volume of 8 ml / kg in pressurecontrolled mode, while in the V group, the tidal volume was set as 8 ml / kg in the volume-controlled mode. In both groups, the initial respiratory frequency was 12 breaths/minutes, the inspiration/expiration time ratio was 1/2, FiO 2 was 40%, and positive end expiratory pressure (PEEP) was 5cm/H 2 O. While applying mechanical ventilation in all patients, it was aimed to keep the EtCO 2 value between 33-35 mmHg. If the EtCO 2 was above 35 mmhg, the respiratory frequency was primarily increased by 2 units every ve minutes in both groups. In this increase, the frequency was accepted as the upper limit of 18 breaths/minute. If the EtCO 2 values did not decrease under 35 mmHg at the 5th minute after reaching 18 breaths per minute, the P insp value of the patients in the P group was increased by 2 cm/H2O every ve minutes as needed. In the V group, the volume settings were increased by 1 ml/kg every ve minutes as needed. The upper limit was determined as 30 cm/H2O for the P group and 10 ml/kg for the V group. Patients whose CO 2 values did not decrease under 35mmhg despite mechanical ventilation with all these upper limit values were excluded from the study by making more complicated changes in mechanical ventilation and insu ation pressures. If EtCO 2 values were below 33 mmHg, in both groups, it was rst reduced to 10 breaths/min, and if there was no increase after ve minutes, P insp values were decreased by 2 cm/H2O every ve minutes in the P group, while the tidal volume was decreased by 1 ml/kg in the V group. However, tidal volume was not allowed to fall below 6 ml/kg in both groups. Demographic data (gender, age, height, weight, and ASA score) as well as operative data (anesthesia, operation, and insu ation duration) were recorded in both groups, T0 was de ned as T0 before anesthesia, T1 after intubation, T2 5 minutes after insu ation, T3 just before desu ation, and T4 5 minutes after desu ation. Heart rate, systolic/diastolic arterial pressure values, saturation of pulseoximetry (SpO 2 ) and NIRS values were recorded at all time points. Additionally, EtCO 2 in T1, T2, T3 and T4; arterial blood gas results for pH, pO 2 , pCO 2 , HCO 3 , BE and Lactate; Tidal volume, respiratory frequency, peak pressure (P peak ), plateau pressure (P plateau ) and, PEEP was recorded. Statistical analysis SPSS 15.0 for Windows program was used for statistical analysis. Descriptive statistics; numbers and percentages for categorical variables, mean, standard deviation, minimum and maximum for numerical variables were given. Comparisons of numerical variables in two independent groups were made using the Student t-Test (when the normal distribution exists), the Mann Whitney U test (when the normal distribution condition was not exist). The rates in the groups were compared with Chi-Square Analysis. Statistical alpha signi cance level was accepted as p<0.05. Results CONSORT diagram of the study was presented in gure 1. In total 70 patients were evaluated in the study between March and July 2020. Groups did not differ for age, BMI, operative time, anesthesia duration and insu ation duration (p>0.05 for all comparisons) ( Table 1). Hemodynamic parameters were presented in Table 3. The systolic, diastolic and, mean arterial pressures did not differ between groups (p>0.05 for all comparisons). The heart rate at T0, T2 and T3 was signi cantly high in group P (p=0,017 p=0,043 p=0,020 respectively). The SpO 2 levels was signi cantly lower in group P at T0 (p=0,006). The EtCO 2 levels was signi cantly higher in group P at T2 time point (p=0,008). Other comparisons for hemodynamic parameters were not signi cant. The comparisons for blood gas parameters were not signi cant as well (p>0.05 for all comparisons) ( Table 4). Discussion In a randomized controlled setting, our results indicate that cerebral oxygenation was better in patients ventilated with PCV mode due to higher NIRS values and lower P peak and P plateau values. The laparoscopic surgery improves the quality of life by avoiding abdominal incisions, extensive dissection and related comorbidities. 1 However pneumoperitoneum causes an increase in intra-abdominal pressure and indirectly a decrease in lung volumes, functional residual capacity and pulmonary compliance. An increase in airway resistance may be resulted with development of atelectasis in the basal parts of the lung and ventilation-perfusion mismatch can occur. 1,3 The VCV mode increases P peak and P plateau values which are directly related with the lung damage. In a randomized controlled setting, Sen et al. compared VCV and PCV on 40 patients who underwent laparoscopic cholecystectomy. The results indicate that P peak and P plateau pressures were higher in patients who underwent VCV after pneumoperitoneum. 7 Netthra et al. compared VCV and PCV on 60 laparoscopic cholecystectomy patients. Their results indicate that PCV resulted with lower P mean and Ppeak values. Our study is also consistent with the studies which resulted in favor of PCV in laparoscopic cholecystectomy. Our results indicate that P peak and P plateau values were found to be signi cantly higher in the VCV group, especially after insu ation. Literary data indicate that VCV may decrease the safety index by increasing the risk of volu-trauma and barotrauma in the VCV mode in laparoscopic cases. To stop the increase in P peak pressure and decrease lung injury, applications such as changing the respiratory rate and tidal volume or switching to PCV mode are performed. 9 Although the PCV mode is a good method in the management of elevated P peak values, its effects on ventilation dynamics and hemodynamic parameters did not clearly de ne. The high P peak values in VCV mode may also resulted with decrease in partial oxygen pressure. However, the effect of VCV and PCV modes on tissue oxygenation are contradictory. Balick-Weber et al. examined the respiratory effects of laparoscopic surgery on 21 patients. No change was shown on partial oxygen pressures after insu ation. 10 Hans et al. also reported no signi cant difference between PO2 pressures on 40 obese patients who underwent laparoscopic by-pass operation. 11 However, in two other studies conducted in obese patients, partial oxygen pressures were shown to be higher in patients ventilated with PCV mode. 12,13 In our study, partial oxygen pressure values were higher in PCV mode, however no signi cant difference was found for blood gas parameters between groups. Tissue oxygenation measurements have been used frequently in perioperative patient management in recent years. Different methods such as bispectral index electroencephalography or auditory evoke potentials are used to measure anesthetic depth. The NIRS is another method which used to evaluate the depth of anesthesia by measuring the oxygenation change at the tissue level in the prefrontal cortex. 14 We could not be able to nd a study that evaluates the cerebral oxygenation with NIRS in laparoscopic surgery. However, NIRS was used in different surgeries previously. Green et al. 6 in their study with 46 patients who underwent major abdominal surgery, detected low tissue oxygenation using the NIRS method, which could not be detected by conventional monitoring methods. Gibson et al. 15 compared NIRS values before and after insu ation in 70 patients who had undergone laparoscopic abdominal surgery and showed that NIRS values decreased statistically after insu ation. Although there was no signi cant difference between SpO 2 and PaO 2 pressures in our study, the NIRS values of patients who underwent PCV were found to be signi cantly higher during pneumoperitoneum compared to the VCV group. This may be an evidence that oxygenation disorder occurs at the tissue level, although the resulting oxygenation change is not re ected in conventional monitoring parameters and arterial blood gas analysis. Kurukahvecioğlu et al. 16 in their study with 60 patients who had undergone laparoscopic abdominal surgery showed that insu ation pressure caused blood to pool in the lower extremities, which decreased cerebral NIRS values. This decrease is a mechanical result of the high pressure created by insu ation in the abdomen. This mechanical condition occurs not only in the abdomen, but also in the thorax, with the high P peak created by the VCV mode, as demonstrated in our study. Increased intrathoracic pressure reduces preload and indirectly cardiac output, and consequently explains the signi cantly lower NIRS values in the VCV group in our study. The limitation of our study is that we had to use P peak and P plateau instead of trans-pulmonary pressure to evaluate the safety of controlled mechanical ventilation modes. Because transpulmonary pressure is the most objective parameter in the evaluation of ventilator induced lung injuries. However, it was not preferred because it is measured by invasive methods. Conclusion In laparoscopic cholecystectomy operations, tissue oxygenation with PCV mode is higher than with VCV mode. In PCV mode, the risk of lung barotrauma, which is likely due to high P peak and P plateau values, is lower. NIRS can be used in laparoscopic cholecystectomy cases because it is more sensitive, noninvasive and easy to use than arterial blood gas analysis in measuring tissue oxygenation. Availability of data and materials: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.	2021-10-24T15:16:05.804Z	2021-10-22T00:00:00.000	{ "year": 2021, "sha1": "dbb39ed6785f5d685fb5bfde808cc007e1433dbf", "oa_license": "CCBY", "oa_url": "https://www.researchsquare.com/article/rs-871798/v1.pdf?c=1637224155000", "oa_status": "GREEN", "pdf_src": "MergedPDFExtraction", "pdf_hash": "1aa52598b669ee69becf162910085223cbb3050e", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
5016361	pes2o/s2orc	v3-fos-license	Modeling and Analysis of Unsteady Axisymmetric Squeezing Fluid Flow through Porous Medium Channel with Slip Boundary The aim of this article is to model and analyze an unsteady axisymmetric flow of non-conducting, Newtonian fluid squeezed between two circular plates passing through porous medium channel with slip boundary condition. A single fourth order nonlinear ordinary differential equation is obtained using similarity transformation. The resulting boundary value problem is solved using Homotopy Perturbation Method (HPM) and fourth order Explicit Runge Kutta Method (RK4). Convergence of HPM solution is verified by obtaining various order approximate solutions along with absolute residuals. Validity of HPM solution is confirmed by comparing analytical and numerical solutions. Furthermore, the effects of various dimensionless parameters on the longitudinal and normal velocity profiles are studied graphically. Introduction The interest in behavior of fluid flow through porous media began in the early days of oil and gas production, where the focus was on estimating and optimizing production. Similarly, another important application is the simulation of ground water pollution, mostly occurring due to leakage of chemicals from tanks and oil pipelines. The objective is to consider groundwater as one medium and polluted water as another, so that the spreading in the latter medium and its consequences can be studied. In recent times, after the introduction of the modified Darcy Law [1], analysis through porous medium has been an important topic for the research community, as it finds is use in fields such as reservoir, petroleum, chemical, civil, environmental, agricultural, and biomedical engineering. Some practical applications in these fields include chemical reactors, filtration, geothermal reservoirs, ground water hydrology, drainage and recovery of crude oil from pores of reservoir rocks [2][3][4][5][6][7]. Squeezing flow has attracted significant attention because of its broad applications in many fields such as chemical, mechanical, and industrial engineering, and in bio-mechanics and food industries. Practical applications of squeezing flows in these fields are polymer processing, modeling of lubrication systems, and compression and injection molding, etc. These flows are induced by applying normal stresses or vertical velocities by means of a moving boundary, which can be frequently observed in various hydro-dynamical tools and machines. Pioneering work on squeezing flows was investigated by Stefan [8] in which he proposed an adhoc asymptotic solution of Newtonian fluid. A solution considering inertial terms was found by Thorp [9]. However, Gupta and Gupta [10] later showed that this solution failed to satisfy boundary conditions. The effect of the inertial term in squeezing films between circular plates has been evaluated by Kuzma [11]. Elkouh [12] studied the squeeze film between two plane annuli taking fluid inertia effects under consideration. Verma [13] and Singh et al. [14] set up numerical solutions of the squeezing flows between parallel plates. Leider and Bird [15] carried out theoretical analysis for squeezing flow of power-law fluid between parallel plates. Naduvinamani et al. [16] investigated squeeze film lubrication of a short porous journal with couple stress fluids. Steady axisymmetric squeezing fluid flow in a porous medium has been analyzed by Islam et al. [17]. Hamza [18] worked on squeeze films considering MHD effect. Suction and injection effects on the flow of electrically conducting viscous fluid squeezed between two parallel disks was studied by Domairry et al. [19]. The study of the porosity and squeezing effects, while investigating the unsteady squeezing flow of visco-elastic Jeffery fluid between parallel disks, has been performed by Qayyum et al. [20]. Apart from the mentioned scholars, other researchers have also carried out different theoretical and experimental studies of squeezing flows [21][22][23][24]. No-slip boundary condition is one of the main concepts of fluid dynamics. Consider a liquid flowing over a solid wall. The condition in which the liquid molecules near the solid wall are motionless, relative to the wall, is called no-slip boundary [25]. This boundary condition has been employed in modeling various viscous and visco-elastic fluid flow problems. Firstly, Navier [26] proposed the general boundary condition which shows fluid slip at the liquid-solid interface. According to him, the difference between the boundary and fluid velocities is proportional to the shear stress at the boundary. The dimension of proportionality constant is length, and this is known as the slip parameter. There are numerous situations in which no-slip boundary condition is not appropriate. For instance, flow on multiple interfaces, polymeric liquids when the weight of the molecules is high, fluids containing concerted suspensions, and thin film problems. A number of perturbation techniques which can solve non-linear boundary value problems analytically are discussed in literature. But the assumption of small parameter is a limitation in these techniques. Recently, a technique was proposed by He [27][28][29][30], that combines homotopy and the traditional perturbation method [31][32][33][34]. This technique was the beginning of homotopy perturbation method (HPM). In a series of papers, He applied this method to discuss nonlinear boundary value problems [27][28][29][30]. As a result, many researchers have used HPM to solve non-linear differential equations in different fields as it is not only easy to use, but also successful. This method minimizes the limitations commonly associated with perturbation techniques, while taking full advantage of the traditional perturbation methods. In fluid dynamics, Siddiqui et al. [35,36] applied this technique for solving non-linear boundary value problems arising in Newtonian and non-Newtonian fluids. In addition, Zhou and Wu [37] used this technique in an inverse heat problem. Also, Hamid et al. [38] compared the method with other analytical and numerical techniques, while solving higher order non-linear differential equations. The objective of this manuscript is to use HPM for the solution of an unsteady axisymmetric squeezing fluid flow between two circular plates through porous medium with slip boundary condition. Validity of HPM solution is confirmed by comparing analytical and numerical solutions. In addition, effects of different dimensionless parameters on the velocity profiles are studied graphically. Description of the Problem An unsteady axisymmetric squeezing flow of incompressible first grade fluid with density ρ, viscosity μ, and kinematic viscosityu, squeezed between two circular plates having speed v(t) and passing through porous medium channel is considered. It is assumed that at any time t, the distance between the two circular plates is 2h(t). Also, it is assumed that r-axis is the central axis of the channel while z-axis is taken normal to it. Plates move symmetrically with respect to the central axis z = 0 while the flow is axisymmetric about r = 0. The longitudinal and normal velocity components in radial and axial directions are w r (r,z,t) and w z (r,z,t) respectively. The geometrical representation of the flow is illustrated in Fig. 1. Problem Formulation The basic governing equations of motion are where and W is the velocity vector, p is the pressure, f is the body force, T is the Cauchy stress tensor, A is the Rivlin-Ericksen tensor, μ is the coefficient of viscosity, andr is the Darcy's resistance. According to Breugem equation [39],rcan be written as: where k is the permeability constant. Now, we formulate the unsteady two-dimensional flow through porous medium. After neglecting body force we assume that W ¼ ½w r ðr; z; tÞ; 0; w z ðr; z; tÞ ð6Þ and introduce the vorticity function O (r,z,t)and generalized pressure _ Pðr; z; tÞas Equations (1) and (2) can then be reduced to The boundary conditions on w r (r,z,t) and w z (r,z,t) are where v t ð Þ ¼ dh dt is the velocity of the plates. The boundary conditions in (12) are due to slip at the upper plate when z = h and symmetry at z = 0. If we launch the dimensionless parameter Equations (7), (9), (10)and (11)are converted to The boundary conditions on w r and w z are After eliminating the _ Pðr; z; tÞ between (16) and (17), we obtain: where 5 2 is the Laplacian operator. Defining velocity components as [11] we see that (15) is identically satisfied and therefore, (19) becomes Both R and Q are functions of time but for similarity solution we consider R and Q constants. Sincev ¼ dh dt , integrating the first equation of (22), we obtain: where C and D are constants. When C>0 and D>0, the plates move away from each other symmetrically with respect to x: The squeezing flow exists when the plates approach each other when C>0, D>0 and h(t)>0. From (22) and (23) it follows that Q = -R. Then (21) becomes After using (18) and (20), we establish the following boundary conditions in case of slip at the upper plate: Fundamental Theory of HPM [27][28][29][30] To exhibit the basic theory of HPM, let us consider the following differential equation: where w is an unknown function and g(r) is a known function. L, N, B are linear, nonlinear and boundary operators respectively. Also U is the boundry of the domain O. We construct Homotopy yðr; pÞ : O Â ½0; 1 ! R which satisfies where p ε [0, 1] is an embedding parameter, and w 0 is the initial guess of (26) which satisfies the boundary conditions. From (27), we have: Thus, as p varies from 0 to1, the solution θ(r,p) approaches from w 0 (r) towðrÞ. Results and Discussions In the present article, we considered an unsteady axisymmetric squeezing flow of incompressible Newtonian fluid passing through porous medium with slip boundary condition. The resulting non-linear boundary value problem is solved through HPM and RK4. There are three parameters; Reynolds number R, constant containing permeability M, and slip parameter γ in the current problem. We present our discussion of results based on different compositions of these parameters. First of all we solve the problem for various values of R; M and ganalytically using HPM. This is illustrated in Tables 1, 2, and 3. Secondly, we solve the problem numerically using RK4 for various R,M and γ. This is explained in Tables 4, 5, and 6. We also check the convergence of HPM solution using different order approximations in Table 7. Finally, we check the validity of HPM solutions by comparing analytical and numerical solutions. This is demonstrated in S1, S2 and S3 Table. All the tables signify the efficiency of HPM. Furthermore, we investigated the effects of various dimensionless parameters on the normal and longitudinal velocity profiles graphically. We show the convergence of HPM solution in Fig. 2. This plot represents the average absolute residuals against different order approximations and it is clearly seen that HPM solution is convergent. Validity of HPM solution is shown in Fig. 3, where we compare HPM and RK4 solutions for fixed values of R, M and γ, and observed that HPM solution is in high agreement with RK4 solution. The effect of the Reynolds number R on velocity profiles is shown in Fig. 4. In these profiles we varied R as R = 0.5,1,1.5,2 and observed that the normal velocity decreases with an increase in R. Also, the longitudinal velocity decreases near the central axis of the channel and increases near the plates. It has been analyzed that the normal velocity monotonically increases while longitudinal velocity monotonically decreases from ξ = 1 to ξ = 1 for fixed positive value of R at a given time. The effect of γ on the velocity profiles is depicted in Fig. 6. In these profiles we varied γ as γ = 0.8,1,1.5,3 and noted that normal velocity increases with the increase in γ whereas longitudinal velocity increases near the central axis of the channel and decreases near the plates. The effect of R = M on the velocity profile is given in Fig. 7. In these profiles, we see that the normal velocity decreases with the increase in R = M while longitudinal velocity increases near the wall and decreases near the central axis of the channel. S1, S2 and S3 Figs. depict the effects of M = γ, R = γ and R = M = γ on the velocity profiles respectively. In these profiles, we observed that normal velocity increases with the increase in M = γ, R = γ and R = M = γ respectively while longitudinal velocity decreases near the wall and increases near the central axis of the channel. It can be observed from these profiles that similar behavior of normal and longitudinal velocity has been captured when we vary M,γ,R = γ,M = γ and R = M = γ while keeping other parameters fixed. It is also observed that R and R = M have a similar effect on the normal and longitudinal velocity profiles while keeping other parameters fixed. Conclusions In this article, we find the similarity solution for an unsteady axisymmetric squeezing flow of incompressible Newtonian fluid through porous medium with slip boundary condition using HPM analytically and RK4 numerically. We determined the convergence of HPM solution using various order approximate solutions. In addition, we checked the validity of HPM solution by comparing analytical and numerical solutions. We observed some key findings related to the effects of dimensionless parameters on the velocity profiles. It was found that: • The normal velocity decreases with the increase in Reynolds number R. • With the increase in Reynolds number R, longitudinal velocity increases near the walls and decreases near the central axis of the channel. • The normal velocity monotonically increases and the longitudinal velocity monotonically decreases from ξ = 0 to ξ = 1 for fixed positive value of R at any given time.	2018-04-03T04:09:19.151Z	2015-03-04T00:00:00.000	{ "year": 2015, "sha1": "95b437cc6b56def3f7e64fc4d12834bf6da0adac", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0117368&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "95b437cc6b56def3f7e64fc4d12834bf6da0adac", "s2fieldsofstudy": [ "Engineering", "Environmental Science" ], "extfieldsofstudy": [ "Medicine", "Physics" ] }
234177824	pes2o/s2orc	v3-fos-license	Characterization of Environmental Covariates of Coimbatore District using Principal Component Analysis The environmental covariates are a key approach in spatial prediction of soil properties which represent the soil forming factors. The environmental covariates are classified into five categories based on CLORPT model, namely climate, organism, relief or topography and parent material (14) and these variables are briefly described in Table 1. Among all categories, climate is the most important factor. Topography tends to be a passive factor for soil formation, as it has a major influence on the soil distribution and vegetation. Each category has different set of environmental covariates. It is very difficult to predict the results with larger set of environmental covariates. To identify the most influencing environmental covariates, principal component analysis (PCA) is used (4). PCA is used for different purposes such as interpreting and visualizing data, finding interrelations between variables in the data, decreasing the number of variables for making further analysis simpler (3) and for many other similar reasons. PCA is a very International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 10 Number 01 (2021) Journal homepage: http://www.ijcmas.com Introduction The environmental covariates are a key approach in spatial prediction of soil properties which represent the soil forming factors. The environmental covariates are classified into five categories based on CLORPT model, namely climate, organism, relief or topography and parent material (14) and these variables are briefly described in Table 1. Among all categories, climate is the most important factor. Topography tends to be a passive factor for soil formation, as it has a major influence on the soil distribution and vegetation. Each category has different set of environmental covariates. It is very difficult to predict the results with larger set of environmental covariates. To identify the most influencing environmental covariates, principal component analysis (PCA) is used (4). PCA is used for different purposes such as interpreting and visualizing data, finding interrelations between variables in the data, decreasing the number of variables for making further analysis simpler (3) and for many other similar reasons. PCA is a very The principal component analysis (PCA) is used to identify the most influencing variable. It is one of the statistical techniques for reducing the dimension of the data. The study was conducted in Coimbatore district, Tamil Nadu with 340 profile points. More than 30 environmental covariates are available for this analysis. To make the analysis easier and accurate the data has to be reduced. The principal components (PC1, PC2, PC3 and PC4) are selected for further analysis which accounts for 53.84% of variation. From the selected four principal components the variables which are having higher percentage of variation were identified. Hence it is one of the easiest methods to predict the most influencing variable using R software. versatile method that enables an interpretation of datasets that can include, for example, multilinearity, missing values, categorical data, and imprecise measurements. "PCA was first coined by Pearson (1901), and developed independently by Hotelling (1933)". It is one of the methods for reducing variables without much loss of information. The main use of principal component analysis (PCA) is to define trends in the data and to guide the data by emphasizing their similarities and differences. Materials and Methods The study was conducted in Coimbatore district of Tamil Nadu located between 11°24'23" to 10°13'12" N Latitude and 76°39'20" to 77°18'00" E longitude with an area of about 4721.28 sq.km as shown in Figure 1. Geomorphology, Physiography, Western Ghats, Geology, ACZ and AEZ are in shape file format (Polygon feature). Hence, these variables are converted to raster format using Feature to Raster tool in ArcGIS software. The extracted environmental covariate includes remotely sensed spectral data and derivatives of terrain attributes. Totally 33 terrain attributes were layer stacked using R software. In order to indentify the most influencing variables, the selected 340 points with corresponding 33 layer staked variables are used to run the principal component analysis (PCA) in R software. Principal component analysis Principal component analysis (PCA) is a statistical tool for dimensional reduction that is often used by converting a large set of variables into a smaller one that also includes much of the information in the larger set to reduce the dimensionality of larger data sets. Principal component analysis (PCA) uses single value decomposition (SVD) to reduce the dimension of the data. PCA is derived from the decomposition of a covariance or a matrix of correlation. It uses orthogonal transformation (15) to translate a set of measurements of potentially associated variables into a set of values of linearly uncorrelated variables called principal components, Principal components are the linear combinations of the initial variables that are squeezed to contain maximum information in the first principal component. The first few principal components hold maximum variability of the model. The second and third component explains less variation compared to first component. These are the uncorrelated variable by discarding the component containing the lowest information. By ranking the eigenvector of the covariance matrix that explains the variation of the principal components gives the order of significance. This analysis was used on the environmental covariates to reduce the dimension and to identify the most influencing variable of the data. Steps involved in Principal Component Analysis (PCA) Step 1: Standardization of the dataset. The Principal Component Analysis initially standardizes the data to remove the scale difference between the variables and convert to z values. Scaling is done to remove the difference in their range of the variable that affects the performance of the analysis. Mean is computed by dividing sum of the observations with the number of observations. Let x 1 , x 2 , x 3 ,…, x n be the number of observations. The mean is calculated by Standard Deviation is the best measure of dispersion. It is calculated from mean of squared deviation of individual values from their mean. It is always positive ranges from zero to infinity. Step 2: Calculation of covariance matrix for the features in the dataset The analysis computes the covariance (pxq) symmetric matrix that explains the correlation of the variables where p is the number of dimensions and q is the number of variables. The covariance matrix was calculated using the matrix equation Where is the mean vector, Step 3: Calculation of eigenvalues and eigenvectors for covariance matrix. The eigenvectors of the covariance matrix are referred as principal components (5). The eigenvectors and eigenvalues are constructed from the covariance matrix. They explain the percentage of variation of each principal component. Where is eigenvalue, v is eigenvector, A is square matrix and det is the determinant of the matrix. The eigenvectors of a matrix are perpendicular to each other. The eigenvectors provides the information about the pattern of the given data. Step 4: Picking k eigenvalues and formation of matrix of eigenvectors. For n variable, there will be n eigenvalues and eigenvectors. The eigenvalues are ordered from largest to smallest to select the components in the order of significance. The eigenvalues likely to be greater the one are selected to form the principal components which explains the maximum variation. Mostly first k eigen values (7) are selected to reduce the dimension of the data. Step 5: Transformation of the original matrix The data has to be transformed by multiplying the k eigenvectors with feature matrix. The feature matrix is a matrix of vectors. The principal component can be selected based on the scree plot (13), where X axis represent the principal components, Y axis represents the variations explained by each component. The scree plot shows the variation captured by each principal component. In Scree plot, the knee point or bending point shows the number of principal component to be selected. An eigenvalue greater than one indicates that PCs account for more variance than accounted by one of the original variables in standardized data. This is commonly used as a cutoff point for which PCs are retained. When the eigenvectors are plotted in scatter plot, the principal eigenvectors fits well with the data. The loading plot shows how each variable characterizes the principal component. The PCA plot shows the clusters of samples based on their similarities. Results and Discussion The proportion of variation explained by each eigenvalue is given in the second column in table 2. It shows that the first nine principal components represent an eigenvalues of more than one which accounts for about 74.7% of variation. The first component accounts for maximum total variation as possible, the second component accounts for the remaining variation. The first principal component explains 22.58% variation followed by second, third and fourth component explains 15.72, 9.772 and 5.769 respectively. The scree plot shows the proportion of information retained by each principal component (Figure 2). The bend or knee point in scree plot indicated that the first four principal component was selected for further analysis. The first four principal components account for 53.8% of variation.	2021-05-11T00:03:30.764Z	2021-01-20T00:00:00.000	{ "year": 2021, "sha1": "ec4d99a4a1bbf01464eb6df31244005859accb5a", "oa_license": null, "oa_url": "https://www.ijcmas.com/10-1-2021/R.%20Priyadharshini,%20et%20al.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "49223545c901191b904aa897d707e0a83d2bb16e", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Mathematics" ] }
249700479	pes2o/s2orc	v3-fos-license	Research ethics to consider when collecting oral histories in wilderness areas such as the Kruger National Park 61 of 2003 and Health Professions Council of South Africa of 2016, which stipulates that an accredited research ethics committee must approve all research involving human participants (Denis 2008:63). The University of South Africa (Unisa) requires all researchers to obtain ethical clearance, particularly when the study involves human participants. Research ethics should involve the following criteria: researchers should ensure that projects adhere to values and principles determined by the research ethics committee. Any adverse circumstances arising in the undertaking of the research project relevant to the ethicality of the study should be communicated. Researchers should conduct the study according to methods and procedures determined to be just and that do not place participants in unnecessary harm. In the last half century, oral history has emerged as a historical approach that is being considered by archivists involved with the collection and accessibility of archival collections for researchers and interested members of the public. The approach to ethics by oral historians has emerged from two major fears: the fear of failing as researchers and the fear of failing the narrators and doing harm. Archivists also need to be cognisant of these fears when collecting oral history. Confronting these fears makes it possible to understand the complex questions behind oral historians’ and archivists’ preoccupations and sheds light on how oral history has evolved and expanded as a field. The research objectives of this article are to determine the three principles identified from the Belmont Report that relate and should be applied to the collection of oral histories by archivists and historians from communities and individuals residing and working in and alongside the Kruger National Park. The theoretical framework for this article is the critical race theory to address historical accounts from communities and individuals sidelined by the mainstream media in South Africa. For the purposes of this article, the study was conducted with the Makuleka and Tsonga communities to determine what ethical implications need to be respected when conducting oral history projects with communities. Contribution: This article will contribute to ethics concerning social sciences and specifically the collection of oral history. Introduction Since the late 1940s oral history has been established as an academic discipline, its methodology is constantly being refined and its theoretical assumptions are questioned in South Africa and the rest of the world (Denis & Ntsimane 2008:64). Oral history remains eminently contextual as it raises issues during interviews and in interactions that occur during interviews and the manner in which communities make sense of the memories being collected (Denis & Ntsimane 2008:65). Oral history has emerged as a historical approach that is being considered by archivists involved with the collection and accessibility of archival materials for researchers and interested members of the public. The approach to ethics by oral historians has emerged from two major fears: the fear of failing as researchers and the fear of failing the narrators and doing harm. Archivists also need to be cognisant of these fears when collecting oral history. The main aim of research ethics is to protect the welfare of the research participants. Social science research institutions in South Africa are required to comply with ethical guidelines as determined by the Health Act, no. 61 of 2003 and Health Professions Council of South Africa of 2016, which stipulates that an accredited research ethics committee must approve all research involving human participants (Denis 2008:63). The University of South Africa (Unisa) requires all researchers to obtain ethical clearance, particularly when the study involves human participants. Research ethics should involve the following criteria: researchers should ensure that projects adhere to values and principles determined by the research ethics committee. Any adverse circumstances arising in the undertaking of the research project relevant to the ethicality of the study should be communicated. Researchers should conduct the study according to methods and procedures determined to be just and that do not place participants in unnecessary harm. In the last half century, oral history has emerged as a historical approach that is being considered by archivists involved with the collection and accessibility of archival collections for researchers and interested members of the public. The approach to ethics by oral historians has emerged from two major fears: the fear of failing as researchers and the fear of failing the narrators and doing harm. Archivists also need to be cognisant of these fears when collecting oral history. Confronting these fears makes it possible to understand the complex questions behind oral historians' and archivists' preoccupations and sheds light on how oral history has evolved and expanded as a field. The research objectives of this article are to determine the three principles identified from the Belmont Report that relate and should be applied to the collection of oral histories by archivists and historians from communities and individuals residing and working in and alongside the Kruger National Park. The theoretical framework for this article is the critical race theory to address historical accounts from communities and individuals sidelined by the mainstream media in South Africa. For the purposes of this article, the study was conducted with the Makuleka and Tsonga communities to determine what ethical implications need to be respected when conducting oral history projects with communities. Researchers should ensure that the research project will adhere to any applicable national legislation, professional codes of conduct, institutional guidelines and scientific standards relevant to the specific field of study. In South Africa, researchers must consider the Protection of Personal Information Act, no. 4 of 2013, the Children's Act, no. 38 of 2005and the National Health Act, no. 61 of 2003(Unisa 2016. Conducting oral history in South Africa requires researchers, both historians and archivists, to consider the principles of ubuntu. By doing so, this will create a moral theory grounded on the concept of human dignity. In accordance with this moral theory, human beings have dignity by virtue of their capacity for community and the combination of identifying with others and exhibiting solidarity with them, particularly where human rights violations are egregious degradations (Metz 2010). Metz (2010) constructed an ethical principle that grows out of indigenous understandings of ubuntu, which accounts for the importance of individual liberty and which is applicable in present-day South Africa. According to Metz (2010:83-85), 'a person is a person through other persons', and this article will discuss what ethical considerations should be considered when undertaking oral history projects. This article will specifically focus on two projects undertaken in and near the Kruger National Park, involving individual persons and communities within these locations. The two locations selected are the geographical area between the Luvuvhu and Limpopo rivers and the Timbavati area on the mid-western border of the Kruger National Park. Problem statement According to Field (2012), oral history is regarded as a method of collecting narratives from individuals and communities whose histories have been neglected by previous dispensations. 'History is always in transit, even if periods, places or professions sometimes achieve relative stabilisation' (Field 2012:3-4). The concept of oral history is a method that creates its own documents that are explicit dialogues about memory (Field 2012). Oral history texts are created, unlike artefacts that are collected. Thus, oral history texts that have gone through archival processes should be referred to as 'oral history collections' (Field 2012:4). In South Africa, there is a dire need to collect oral histories from communities and individuals from various locations in South Africa whose narratives were sidelined by the apartheid dispensation. The focus of this research is the oral history narratives that relate to wilderness areas in South Africa, and the cultural significance these areas have for communities and individuals that have lived and worked there. Ethics is a fundamental concern and should always be observed when collecting oral history, particularly when it involves individuals and communities that may feel their information and knowledge may be exploited. Ethics should be observed by historians and archivists when collecting oral history. Archivists and historians should observe the Belmont Report on Ethics, which states a principle of autonomy that requires decision-making capacities of research participants as autonomous persons that should be respected by researchers (Francis, Rakotsoane & Nicolaides 2019:17-20). This means that research participants must participate freely in any research without any controlling influences that would mitigate against a free and voluntary act (Francis et al. 2019). The second principle that should be observed is nonmaleficence, which requires researchers to not intentionally create harm or injury to the research participants. This principle affirms the need for professional competence and articulates commitment from the researchers to protect their research participants (Beauchamp & Childress 2013). The third principle involving research participants is that of justice, which requires researchers to distribute benefits, risks and costs fairly among all parties involved. This principle ensures that people's rights and their acceptable laws are respected, particularly in communities where poverty, illiteracy and non-availability of regulatory frames are the order of the day (Francis et al. 2019:22-25). These principles will be discussed in relation to the collection of oral histories pertaining to communities and individuals residing and working in the Kruger National Park. Research methodology A qualitative approach was used to collect and analyse oral histories related to two communities adjacent to the Kruger National Park. These areas are the Luvuvhu area in Northern Kruger and the Timbavati area in the central western area of Kruger. A qualitative research method was deemed appropriate as in the analyses of historical data and the application of ethics when conducting oral history (Leedy & Ormrod 2014:141). Research objectives and questions The research objectives of this article were to determine how the three principles identified from the Belmont Report (Visagie, Beyer & Wessels 2019) relate and should be applied to the collection of oral histories by archivists and historians from communities and individuals residing and working in and alongside the Kruger National Park, in Mpumalanga and Limpopo provinces in South Africa. It will also share information relating to a few historical sites within the Kruger National Park and the oral history projects that have been undertaken to collect such information from individuals and communities. Theoretical framework The theoretical framework of this article is the critical race theory. The purpose of this theory is to address historical accounts from communities and individuals that have been sidelined by mainstream media in South Africa. The histories of individuals and communities contribute to the history of the Kruger National Park and by all accounts should be allowed to be heard and shared by interested individuals. According to American scholar Lintner (2004), critical race theory is a framework or set of basic perceptions, methods and pedagogy that seeks to identify, analyse and transform structural and cultural aspects of society that maintain the subordination and marginalisation of groups of people (Lintner 2004:27-32). Furthermore, critical race theory focuses on challenging the dominant discourses on race and racism with particular reference to legal systems (Lintner 2004). However, it is the view of the author of this article that critical race theory can also be applied to the collection of oral history on the African continent, in general, and South Africa, in particular, thereby contributing to the decolonisation of South African history. Although critical race theory has been applied to historical studies in the United States, it also has relevance in South Africa and can be a theory that is the basis for the concepts of decolonisation of South African history. The relationship between critical race theory and the South African position of arguing for the decolonisation of South African history implies that historians and archivists need to become cognisant of not professing a set of social, economic and political privileges that may manifest into biases or stereotypes (Lawrence 1997). Examination of biases of one's own action requires that archivists and historians attempt not to incorporate their personal prejudices (Gewinner, Krohn & White 2000:113). Critical race theory has four major themes. Firstly, race and racism are timeless and endemic and permanently intertwined in the South African social fabric. Secondly, this theory seeks to challenge constructed ideologies objectivity and racial sensitivity and argues that such constructs are shelters for hegemonic practices by dominant groups in South Africa. Thirdly, critical race theory is committed to social justice and eradication of social subjugation. And, finally, critical race theory seeks to promote experiential knowledge of women and disenfranchised people as legitimate to the understanding of subjugated people (Solorzano 1997). Both critical race theory and decolonisation are theories apt in a postmodernist setting. According to Derrida (1996) and Foucault (1972), the postmodernist theory stipulates that the archive is linked to storytelling and the archive is constructed in a creative form. Postmodernism implies that the archivists deliberately select information to formulate a particular narrative (Derrida 2001). In other words, both critical race theory and decolonisation are concepts that involve storytelling and the shaping of narratives collected by historians and archivists to determine metanarratives. Historians and archivists select information and accounts to specific events to reveal narratives that they deem worthy of being collected and disseminated. In present-day South Africa, the current metanarrative determined by archivists and historians tends to be about events that occurred and were sidelined by the apartheid dispensation. Although such narratives are collected by the public archivists and their counterparts in organisations, like the South African National Parks Board and the National Film, Video and Sound Archives, the dissemination of these collections to researchers and interested members of the public is poorly executed. Formal ethical guidelines play an important role in regulating research practices and are implicit in regulating research practices and daily relations and engagements fundamental to the research process. This article proposes the need for a move towards an Africanist and decolonial ethical practice that acknowledges that the African continent has vast cultures, traditions and beliefs that have been marginalised by Euro-Western ways of viewing and engaging with the world (Molyneux & Geissler 2008:688;Molobela 2017;Visagie et al. 2019). Socially responsible ethics are decolonial ethics that speak about the importance of acknowledging spaces people occupy and the knowledge they carry. In order to decolonise ethics, it is necessary to acknowledge the multitudes of understanding the world and to see community members as persons with lived experiences who contributed to how they engage with each other and their environment (Molyneux & Geissler 2008). When conducting oral history interviews, it is important that researchers avoid further harm to the historically oppressed by providing them with space to revive and recuperate their culture, history, language and identity by allowing women, the elderly, disabled and children to define themselves and their reality and what can be spoken and written about them (Chilisa 2011;Visagie et al. 2019). Literature review Background and contextualisation of oral history in the Kruger National Park The conservation of wildlife and the preservation of such sites and histories in Africa have largely been formed in relation to the pursuits of livestock management and mining. This is particularly relevant in the case of South African game reserves and their immediate surrounds, often referred to as conservancies or concession areas. Experiences of local communities in relation to the management of the livestock, mining pursuits and interaction with different fauna and flora have largely been neglected in the historical discourse, particularly relating to the Kruger National Park. Researchers such as Jane Carruthers (1995Carruthers ( , 2001Carruthers ( , 2017 and Jacob Dlamini (2020) have written on the socio-political and socio-economic matters associated with the Kruger National Park; however, there are still many areas that remain unexplored. This article's main focus is on the areas of the Pafuri and Timbavti and on the cultural and historical sites that exist in these areas. These are located both in the borders of the Kruger National Park and in the concession areas occupied by communities, as well as hunting and other conservation entities. According to Tucker (2010), some of these sites and corresponding narratives found in the Timbavati area have correlations between the pyramids and the sphinx of ancient Egypt. The earliest archival records that exist are those of James Stevenson-Hamilton related to the origins of the Kruger National Park and resulted in the removal of many communities who were residing within the borders of the game reserve (Stevenson-Hamilton 1993). These removals prevented many communities, like those in the Phabeni http://www.hts.org.za Open Access area, from accessing burial sites of their elders and family members. With the advent of a democratic dispensation in South Africa in the 1990s, the fence between the Kruger National Park and Mozambique was dropped. This development allowed more opportunities for wildlife species such as elephants and other species, to roam more freely so that the Kruger National Park could avoid using controversial methods of culling to control the numbers of elephants and other animals. Although the creation of the Peace Parks was viewed as a milestone in the conservation of this region, the removal of the fence also saw an escalation in the incidents of poaching (Schellnack-Kelly 2017). From the 1990s, endeavours to include local communities in the conservation efforts of the Kruger National Park have been undertaken and efforts have been made by individuals to ensure local, neighbouring communities are included in the conservation projects, with many family members employed by South African National Parks as rangers and guides (Schellnack-Kelly 2017). More could be done to incorporate family members and allow them the opportunity to share their histories and cultural significances that they attach to particular areas and specific animals and plants. It was stated in a News24 media report in May 2017 that the Kruger National Park has 627 cultural heritage and historical sites within the park. Many of these are unknown to members of the public (News24 2017). The preservation of sites, legends and historical narratives of communities that lived within the boundaries and immediate surrounds of the Kruger National Park have been sidelined by the narratives favoured by the colonial and apartheid governments. Thus, the author is determined to uncover these histories and significance of these sites as part of the history of the Kruger National Park. Besides accessing archival collections, the author also believes that oral history collections will provide more substance and incorporate narratives from persons previously excluded from sharing their narratives relating to the Kruger National Park. The approach to ethics by oral historians and archivists has emerged from two major fears: the fear of failing as researchers and the fear of failing the narrators and doing harm. Archivists also need to be cognisant of these fears when collecting oral history. Confronting these fears makes it possible to understand the complex questions behind oral historians' preoccupations and sheds light on how oral history has evolved and expanded as a field (Jessee 2011:287-307). Since 2011, oral history has been celebrated by its practitioners for its humanising potential and its ability to democratise history by bringing the narratives of people and communities typically absent in the archives into conversation with that of the political and intellectual elites who generally write history (Jessee 2011:289). The value of oral history is unquestionable when dealing with the narratives of ordinary people. However, in recent years, oral historians have increasingly expanded their gaze to consider intimate accounts of extreme human experiences, such as narratives of survival. This shift in academic and practical interests begs the question whether there are limits to oral historical methods and theory. Oral history collections and ethical considerations The concept of participant autonomy is central to the ideology and practice of information consent. The root meaning of the concept of autonomy refers to a state of being independent from any external regulations or constraints. Autonomy necessitates a deep respect for people's abilities to decide themselves which laws they wish to comply. An autonomous person has the right to make rational choices, free from external influence and taking personal interest and consequences into consideration (Dworkin 1988:15;Visagie et al. 2019). Furthermore, autonomy is a human right that implies that persons have the right to self-determination, free from undue influences from others, by virtue of their inherent dignity as human beings. The tensions and paradoxes inherent to the view of persons as independent and self-determining find expression in the notion of collective autonomy (Dworkin 1988:12;Visagie et al. 2019). Individual and collective autonomy applied to informed consent in research are complex phenomena and are applicable to the two ethics paradigms of principlism and Afro-communitarianism. These two ethics paradigms imply that it is crucial for researchers when conducting ethical research in rural communities in Africa to question their moral obligations relating to the notion of participant autonomy (Visagie et al. 2019). A person is a dignified being who is able to make independent choices based on a rational assessment of a situation. Principlism has been designed as a standard analytical framework and represents a principle-based, common morality theory that provides a normative structure for ethical analysis and policy design. In addition, Beauchamp and Childress contend that principlism advocates for the consideration of four sets of moral principles that act as norms of obligation (Beauchamp & Childress 2009:14;Visagie et al. 2019). These four interdependent sets of principles are respect for autonomy, beneficence, non-maleficence and justice (Beauchamp & Childress 2009). These moral principles serve to justify moral decisions and should be applied as a framework to inform the formulation of procedural rules as essential to guide archivists and historians when conducting oral history-related activities. Informed consent and its application to oral history collections in South Africa Researchers conducting research in rural communities are faced with challenges in negotiating the balance between individual and collective autonomy. This creates the need for obtaining consent from both individuals and the community. In the absence of published guidelines on the interpretation and application of 'principlist doctrines' on obtaining informed consent, archivists and historians are often unprepared to integrate the customs and values of participants in the informed consent process (Visagie et al. 2019:166-168). Ryen (2016:35) contended that there are three http://www.hts.org.za Open Access main problem areas with the application of Western guidelines for research in Africa. These are consent, trust and confidentiality, which Ryen (2016) regarded as being interlinked areas of consent, securing trust and confidentiality. This scholar further contends that researchers should not include other parties while conducting the research process as this can taint the trust relationship with the research participants and the community (Ryen 2016:40;Visagie et al. 2019). Metz and Gaie (2010) contended that respect for persons is not founded on a narrow view of individual autonomy grounded in Western traditions. We should all strive to live in harmony with others. Collective autonomy veers away from preferences on rational personal choice, liberty and independence. Metz and Gaie (2010) and Visagie et al. (2019) contended that researchers have a moral obligation to consider the common good and act in solidarity with others. These are principles that archivists and historians should consider when collecting oral history narratives from individuals and communities. In essence, the principles of ubuntu are applicable to researchers collecting data and information from both individuals and communities. Discussion and findings relating to the Kruger National Park and surrounding communities Makuleka community -North of Luvuvhu River in the Kruger National Park The Makuleka Contractual Park constitutes the northern most section of the Kruger National Park in South Africa. It comprises approximately 240 km² of land (Wilderness Safaris 2007). The triangle is a wedge of land created by the confluence of the Limpopo and Luvuvhu rivers at the tripoint of Crook's Corner, which forms a border with Zimbabwe along the Limpopo River (Wikipedia 2021). It is a natural wildlife crossing point from North to South and back and is regarded as a distinct ecological region. The region is referred to as the Pafuri, which is a Tsonga word derived from the Mphaphuli, which is the dynastic name of Venda chieftains who ruled over this area (Wikipedia 2021). The Luvuvhu River is named after a tree growing on the banks of this river (Du Plessis 1973:265). From about 1200 AD a large cultural civilisation and trade network began to emerge to the North, with evidence being found as sites such as Mapungubwe, in the northern province of Limpopo. In these areas, sacred leaders were elite members of the community and were believed to have supernatural powers and the ability to predict the future (Wikipedia 2021). The wealth and sophistication of these people is evident by the beautifully crafted jewellery, Arab glass beads and Chinese porcelain found in these sites and accompanying burials of sacred leaders (South African National Parks 2022). The end of Mapungubwe occurred at the same time as the rise of an even greater trading and architectural civilisation, being Great Zimbabwe, which flourished for over a century. In the 1550s, groups of people crossed the Limpopo River and founded numerous settlements in the Pafuri region, including that of Thulamela on the southern bank of the Luvuvhu River (Berger 2005). These walled cities existed in the Pafuri triangle at about the same time that Portuguese trade began on the east coast of South Africa (Mapungubwe 2007;Punt 1976). This Thulamela culture ended around 1650 (Owen 2017 The following information was provided by a spokesperson for the Makuleka people: We, the Makuleka people were one of the first communities to win back our land using South Africa's land restitution laws. We feel that after a series of extraordinary negotiations we have placed ourselves, our supporters and private sector partners at the cutting edge of socially concerned approaches to conservation. The Makuleka Region of the Kruger National Park (KNP) is an attempt to harmonise the protection of biological diversity with our interests as rural people. Up to now we generally despised the notion of conservation, ever since the Kruger Park's first game warden earned the nickname Skukuza (which derives from Shangaan to mean The Sweeper) for the way he forced the indigenous inhabitants out of the park in the early 1900s. We were victims of the same racist approach when we were forced off of our land in 1969. In 1996, we reversed this by creating a Community Property Association, which gained ownership of 22,000 hectares of the northernmost part of the KNP between the Limpopo and Luvhuvu Rivers. The land was returned to us after we reached a mediated settlement with many government departments but most importantly with South African National Parks Board The spokesperson further added that: Instead of poaching on that land for subsistence, our young people are now protecting the wildlife with their own lives. To prove our intent to use conservation and tourism to regenerate the economies of our villages, we added to Kruger another 5,000 hectares of communal land that was never previously incorporated into the park. White lions of Timbavati -Linda Tucker An intriguing narrative surrounds the white lions of the Timbavati. The Tsonga narrative explains how on a particular night, a big star appeared in the heavens as bright as the sun and descended to earth. The star landed in the area of the 'Timbi-le-Vaa-ti' which was ruled by Queen Numbi (Schellnack-Kelly 2017;Tucker 2010). For several years after this event, many of the animals in the Timbavati area gave birth to young animals that were born white with blue eyes. This phenomenon of white lions with blue eyes is a significant feature of these animals. The phenomenon of blue eyes is not only confined to lions but also occurs in leopards. The most well-known was a leopard with blue eyes called Marula, who was frequently sighted by rangers and visitors to the Tanda Tula and Kambuka Safari Lodges, situated in the Timbavati region (Schellnack-Kelly 2017; L. Woodward, pers. Comm., 28 March 2014). The Timbavati is regarded as a sacred region where no hunting is allowed. The San referred to the lions of this area by the name tsau, which means star beasts. The San also regard the white lions to be the children of the Creator, with sangomas believing that the white lions are evidence of snow animals whose thick mane and paw formation are adapted for glacial conditions (McBride 1941;Schellnack-Kelly 2017). The area where the white lions are frequently located are in the 31° longitudinal meridian, which lines up with the Sphinx in Egypt and Great Zimbabwe. In Africa, several cultural sites are located along this meridian, which also lines up with the Leo constellation (Herschel & Lederer 2003). The Timbavati region is an area that is steeped in cultural significance to the communities that live in that area and it must be conserved in order to preserve the cultural significance of these areas to local communities. Conclusion This article aimed to report on a study conducted on both the Makuleka community and Tsonga community to determine what ethical implications need to be respected when conducting oral history projects with communities. Information was gathered from a spokesperson for the Makuleka community, while the information from the Tsonga community was shared by a game ranger working at one of the Timbavati concession areas. The findings of this study focussed on the rights and responsibilities that historians and archivists should observe the following ethical considerations when collecting oral history and disseminating information related to oral history endeavours: 1. Researchers have fundamental rights to academic freedom and freedom of scientific research. 2. Researchers should be competent and accountable. They should act in a responsible manner and strive to achieve the highest possible level of excellence, integrity and scientific quality in their research. 3. Researchers have a right and obligation to refrain from undertaking any research that violates the integrity and validity or compromises their autonomy in research. 4. Researchers have a right and duty to undertake all efforts to bring their research and the findings to the public domain in an appropriate manner. The publishing of research findings should be carried out in a manner that will not harm research participants or their communities. Researchers should be honest with respect to their actions in research and their responses to the actions of other researchers (Unisa 2016). Oral history is essential in fostering an appreciation of incorporating indigenous knowledge, the myths, legends and experiences of all communities as evidence of the necessity to preserve wilderness areas for the benefit of all communities and ensuring their accessibility to all communities. Tucker (2010), Player (1998) and Mutwa (1998) have written extensively on the local communities and indigenous knowledge and the association of these with the environment and sacred places in South Africa. Although the writings of Mutwa probably belong more to the field of anthropology, the information which he provides relating to indigenous culture and the associations of different fauna and flora definitely has an impact on post-apartheid perceptions concerning the environment and local cultures. The influence of Mutwa is also evident at the Nhlapo exhibition hall in Freedom Park in relating the story of creation as revealed in the African culture. This perspective has only begun to receive credence in the past few years and his 1964 narrative Indaba My Children deserves to be part of the South African literary landscape as have Shakespearean, Greek, Roman and biblical narratives been utilised in understanding and moulding Western civilisation perceptions and understanding. The importance of oral histories and indigenous knowledge are crucial to the sustainability of wilderness areas in South Africa. Effective initiatives to capture, preserve and disseminate the history of all South Africans are fundamental to the sustainability of archives and related heritage entities (Schellnack-Kelly & Jiyane 2017:127). In spite of the importance of capturing oral history, it is important that historians and archivists observe ethical principles that do not place any participants or their communities in harm's way. The principles of autonomy and respect for dignity are the essence of three ethical principles: (1) Indigenous people should be recognised as the primary guardians and interpreters of their cultures, arts and science. (2) Indigenous people should be recognised as collective legal owners of their knowledge. (3) The right to learn and use indigenous knowledge can be acquired only in accordance with the laws or customary procedures of the indigenous persons concerned and with their free and informed consent (Denis 2008:68-69). Working with oral history is fundamentally interdisciplinary, bringing together social researchers, anthropologists, historians and archivists. As observed by Thompson and Bornat (2017:390-391), oral history gives history back to the people and also gives people a past. It also assists individuals and communities in forming a future of their own making. Oral history practitioners should see ethical guidelines as a way of doing oral history in a professional manner (Denis 2008:81).	2022-06-16T15:36:21.343Z	2022-05-19T00:00:00.000	{ "year": 2022, "sha1": "73a50257feb8edc9a88efa983d1abdf4edb57d1b", "oa_license": "CCBY", "oa_url": "https://hts.org.za/index.php/hts/article/download/7467/22410", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "0dfad831947624acf88c561cdffd2455b2275f6c", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [] }
4956905	pes2o/s2orc	v3-fos-license	Spontaneously Regressive Angiolymphoid Hyperplasia with Eosinophilia: A Case Report with Evidence of Dendritic Cells Proliferation To the Editor: A 79‐year‐old male presented with multiple asymptomatic, erythema, and nodules on the right‐front scalp for about three weeks. The number of the nodules gradually increased. Physical examination revealed multiple violaceous, reddish nodules measuring 0.5–1.0 cm and infiltrative plaques located on the right frontal scalp, without evidence of lymphadenopathy [Figure 1a]. Serological examination revealed the elevated level of leukocytes. Peripheral eosinophil and serum IgE were normal. Histopathology revealed vascular hyperplasia in the dermis. The larger vessels were lined by characteristic “hobnail” endothelial cells, which protruded into the lumen and have ovoid nuclei and intracytoplasmic vacuoles. There was mixed inflammatory infiltration of prominently eosinophils, histiocytes, lymphocytes, and neutrophils [Figure 1c]. Immunohistochemical examination revealed dendritic cells of epidermis and dermis increased, with positive CD1a staining, the linear density of CD1a positive dendritic cells was 70–80/mm (normal range 42.7 ± 17.9/mm) [Figure 1d]. Based on the history, clinical examination, and histopathology, the patient was diagnosed with angiolymphoid hyperplasia with eosinophilia (ALHE). The patient did not use any topical drug. The lesions regressed spontaneously after one month [Figure 1b]. Correspondence To the Editor: A 79-year-old male presented with multiple asymptomatic, erythema, and nodules on the right-front scalp for about three weeks. The number of the nodules gradually increased. Physical examination revealed multiple violaceous, reddish nodules measuring 0.5-1.0 cm and infiltrative plaques located on the right frontal scalp, without evidence of lymphadenopathy [ Figure 1a]. Serological examination revealed the elevated level of leukocytes. Peripheral eosinophil and serum IgE were normal. Histopathology revealed vascular hyperplasia in the dermis. The larger vessels were lined by characteristic "hobnail" endothelial cells, which protruded into the lumen and have ovoid nuclei and intracytoplasmic vacuoles. There was mixed inflammatory infiltration of prominently eosinophils, histiocytes, lymphocytes, and neutrophils [ Figure 1c]. Immunohistochemical examination revealed dendritic cells of epidermis and dermis increased, with positive CD1a staining, the linear density of CD1a positive dendritic cells was 70-80/mm (normal range 42.7 ± 17.9/mm) [ Figure 1d]. Based on the history, clinical examination, and histopathology, the patient was diagnosed with angiolymphoid hyperplasia with eosinophilia (ALHE). The patient did not use any topical drug. The lesions regressed spontaneously after one month [ Figure 1b]. ALHE is an uncommon, benign disorder that presents as solitary or multiple red-brown dome-shaped papules or nodules occurring most frequently on the head and neck. The disease is idiopathic. Trauma, hyperestrogenemia, infectious agents, atopy, reactive hyperplasia, and benign neoplasia have been implicated in the minority of cases. The pathogenesis of ALHE is still under controversial. Various hypotheses have been put forth, including a reactive process, a neoplastic process, and infectious mechanisms. Kempf et al. [1] postulated that ALHE might present CD4+ T-cell lymph-proliferative disorder, rather than a true vascular lesion. Central to the histology of ALHE is the proliferation of blood vessels of varying sizes lined by plump endothelial cells. Inflammation is the second defining characteristic. Lymphocytes and varying amounts of eosinophils diffusely surround and may infiltrate the blood vessels. Depending on the stage of the lesion, the vascular or inflammatory component may predominate. In the active growing ALHE, the vascular component predominates, whereas in the late stages of the disease lymphocytes become more prominent. [2] ALHE is different from Kimura's disease clinically and histopathologically. The typical presentation of ALHE is papules or nodules, while the Kimura's disease is subcutaneous mass. Histologically, the proliferation of blood vessels was superficial in ALHE, while the Kimura's disease is deeper, florid lymphoid follicles with germinal center formation were usually seen. Moreover, Kimura's disease is a systemic immune-mediated process that commonly presented with eosinophilia, high level of IgE, and may be associated with renal disease. In our patient, the histopathology supports the diagnosis of ALHE. However, the infiltration of inflammatory cells was more prominent, suggesting the disease was in the late stage. A rare character should be pointed out is the infiltration of dendritic cells in the epidermis and dermis, which has been confirmed by immunohistochemistry. This change has not been reported before. Dendritic cells are considered to be one of the major antigen-presenting cells in the skin. The macrophages of dermis have scavenging and phagocytic activities, as well as anti-inflammatory properties that contribute to microbial clearance, skin homeostasis, and wound repair. The predominantly increasing dendritic cells in the epidermis, which stained CD1a, accompanied with multiple histiocytes in deep dermis probably indicated the underlying immunological mechanism. Treatment of ALHE is often pursued to provide symptomatic relief and address cosmetic concerns. Surgical excision is commonly used. Other alternative treatments have been reported with variable levels of success. These treatments include laser therapy, systemic or intralesional corticosteroid injection, cryotherapy, imiquimod, tacrolimus, isotretinoin, radiotherapy, interferon-alpha 2a, anti-interleukin-5 antibody, photodynamic therapy, and methotrexate. Spontaneous resolution has also been reported. Adler et al. [3] conducted a systematic review of the literature, within the 593 cases, spontaneous resolution, occurring alone or after attempted treatment, was reported in only 17 cases (2.9%). In our case, we provide support for a reactive process for ALHE, according to the prominently increasing dendritic cells and histiocytes. Moreover, the short course of the disease and spontaneous regression also reflected this point from the other aspect. We hypothesize that the dendritic cells may be involved in the pathogenesis of ALHE, especially in very early stage. While it is yet to be confirmed by large-scale research, and further work should be done to explain the pathogenesis of the findings. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.	2018-04-27T03:28:05.979Z	2018-04-20T00:00:00.000	{ "year": 2018, "sha1": "34de95aec352485d51b906b4db84c5123cdf0ccf", "oa_license": "CCBYNCSA", "oa_url": "https://doi.org/10.4103/0366-6999.229900", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "34de95aec352485d51b906b4db84c5123cdf0ccf", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
258881660	pes2o/s2orc	v3-fos-license	Donor Lymphocyte Infusion for Relapsed Acute Leukemia or Myelodysplastic Syndrome after Hematopoietic Stem Cell Transplantation: A Single-Institute Retrospective Analysis Objective The prognosis of the patients who relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is poor, and therapeutic options are limited. In the present study, we investigated the efficacy and factors associated with the survival in patients with acute leukemia or myelodysplastic syndrome (MDS) who relapsed following allo-HSCT and were treated with donor lymphocyte infusion (DLI) in real-world practice. Patients Twenty-nine patients with acute myeloid leukemia (21), acute lymphoid leukemia (4) or MDS (4) were enrolled. Eleven patients were diagnosed with hematological relapse, and 18 were diagnosed with molecular or cytogenetic relapse. Results The median injection number and median total number of infused CD3+ T cells were 2 and 5.0×107/kg, respectively. The cumulative incidence of acute graft-versus-host disease (aGVHD) of grade ≥II at 4 months after the initiation of DLI was 31.0%. Extensive chronic graft-versus-host disease (cGVHD) occurred in 3 (10.3%) patients. The overall response rate was 51.7%, including 3 cases of hematological complete remission (CR) and 12 cases of molecular/cytogenetic CR. Cumulative relapse rates at 24 and 60 months following DLI in patients who achieved CR were 21.4% and 30.0%, respectively. The overall survival rates at 1, 2 and 3 years after DLI were 41.4%, 37.9% and 30.3%, respectively. Molecular/cytogenetic relapse, a longer interval from HSCT to relapse, and concomitant chemotherapy with 5-azacytidine (Aza) were significantly associated with a relatively long survival following DLI. Conclusion These results indicated that DLI was beneficial for patients with acute leukemia or MDS who relapsed after allo-HSCT and suggested that DLI in combination with Aza for molecular or cytogenetic relapse might result in favorable outcomes. Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered a curative therapy for patients with various hematological malignancies, including acute leuke-mia.However, relapse is a major cause of treatment failure and remains a challenging issue to address.The incidence of relapse after allo-HSCT increases from 15-20% in the lowrisk group to 30-40% in the high-risk group according to the genetic risk stratification in acute myeloid leukemia (AML) (1).Therapeutic options for relapse include the dis-continuation of immunosuppressive agents, re-induction by chemotherapy, a second round of allo-HSCT and donor lymphocyte infusion (DLI) with or without chemotherapy (2)(3)(4).However, while a second round of allo-HSCT from the same or a different donor may be considered, the mortality rate due to regimen-related toxicity is high, and further recurrence is frequently observed (5).Previous studies have demonstrated that a second round of allo-HSCT or DLI were superior to the discontinuation of immunosuppressive agents and chemotherapy in terms of the survival (2,3).No significant difference in the three-year survival rate has been reported between a second round of allo-HSCT and DLI (4). DLI is conducted with an expectation of enhancing the graft-versus-leukemia (GVL) effect mediated by T cells through the restoration and activation of T cells and the reversal of T cell exhaustion (6).The efficacy of DLI varies according to the type of disease.DLI for patients with chronic myelogenous leukemia (CML) in the chronic phase is highly effective, with a response rate of 80-90%; however, the response rates of CML in blastic crisis, AML, myelodysplastic syndrome (MDS) and acute lymphoid leukemia (ALL) are all below 40% (7,8). Various modifications of conventional DLI have been investigated in order to improve clinical outcomes (7).Preemptive or prophylactic use of DLI in molecular relapse or high-risk AML or MDS has been explored, based on the finding that a lower tumor burden is critical for a favorable outcome (9)(10)(11).A reduction in the risk of relapse and an improvement in the overall survival (OS) compared with therapeutic DLI or no intervention have been demonstrated (12)(13)(14). To investigate real-world practices concerning treatment using DLI in patients with acute leukemia and MDS who relapsed following allo-HSCT, we examined the efficacy and factors associated with the survival in the present study. Patients Twenty-nine patients with acute leukemia or MDS who relapsed following allo-HSCT and were treated with DLI at Sapporo Hokuyu Hospital from 2004 to 2019 were enrolled in the present study.Informed consent was obtained from all patients.This study was approved by the internal review board at Sapporo Hokuyu Hospital. DLI Soon after the relapse, immunosuppressive agents were tapered and discontinued in order to enhance immunity against leukemia.DLI was performed with careful monitoring for the appearance of graft-versus-host disease (GVHD) if the discontinuation of immunosuppressive agents failed.The dose and interval of DLI and concomitant use of chemotherapy were decided at the discretion of each attending physician.Thirteen patients were treated with DLI alone, and sixteen patients were treated with DLI and chemotherapy, including 5-azacytidine (Aza). Definition Hematological relapse was defined as bone marrow (BM) blasts >5%.Molecular relapse was defined as an increase in the Wilms tumor 1 (WT1) value [>50 copies/μg RNA in peripheral blood (PB)] without hematological relapse.Cytogenetic relapse was defined as reappearance of disease-specific chromosomal abnormalities or a decrease in donor chimerism (<95%).Hematological complete remission (CR) was defined as the achievement of a morphologic leukemia-free state (<5% blasts in the bone marrow, no Auer rods, and no evidence of extramedullary disease) and peripheral blood count recovery (absolute neutrophil count >1,000 cells/μL and platelets >100,000 cells/μL in the absence of growth factor treatment) (22).Molecular CR was defined as a decrease in the WT1 value in PB to <50 copies/μg RNA.Cytogenetic CR was defined by the disappearance of chromosomal abnormalities or the appearance of full donor chimerism. The threshold of WT1 positivity was set at 50 copies/μg RNA in the PB according to the manufacture's recommendation (Otsuka Pharmaceutical, Tokyo, Japan).We observed fluctuations in the WT1 values in several patients with molecular relapse, as shown in Supplementary material 1.However, a WT1 value !50 copies/μg RNA was observed temporarily and soon decreased to the normal range in responding patients.Therefore, we deemed molecular CR to be present when WT1 values in the PB were <50 copies/μg RNA in 2 consecutive assays.Conversely, we deemed molecular relapse to be present when WT1 values in the PB were !50 copies/μg RNA in 2 consecutive assays with an increasing trend.Acute GVHD (aGVHD) was graded according to the established criteria (23), and chronic GVHD (cGVHD) was classified based on the classical criteria (24). Endpoints The primary endpoints were CR and the two-year survival rates.The secondary endpoints were the incidence of GVHD following DLI and the factors associated with the response to DLI and the survival. Statistical analyses Fisher's exact test was used to compare categorical variables.The cumulative incidence of GVHD and the relapse Survival curve comparisons were conducted with the logrank test.A univariate analysis of the variables related to the survival was conducted with the log-rank test.A multivariate analysis was not performed due to the small population size.p<0.05 was considered significant. Characteristics of patients Characteristics of patients are shown in Table 1.A total of 29 patients were enrolled (15 men and 14 women) with a median age of 45 (range: 17-61) years old.Patients' diagnoses consisted of AML in 21, ALL in 4 and MDS in 4. The cytogenetic risk stratification in AML according to European Leukemia Net was favorable, intermediate and poor/adverse in 3, 11 and 7 patients, respectively (22).The disease status at allo-HSCT was CR in 11 patients and non-CR in 18 patients.Five patients received BM stem cells from HLAmatched sibling donors, 10 patients received PB stem cells from HLA-matched sibling donors, 6 patients received BM or PB stem cells from HLA-haploidentical sibling donors, and 8 patients received BM stem cells from unrelated donors. The conditioning regimen was myeloabelative (MAC) in 14 and reduced intensity (RIC) in 15.Twelve patients were administered cyclosporine A + short-term methotrexate (MTX), and 11 patients were administered tacrolimus+shortterm MTX for prophylaxis of GVHD.Patients who underwent haplo-HSCT were treated with a conditioning regimen consisting of fludarabine, busulfan and total-body irradiation.Tacrolimus+myccopenolate mofetil and PT-CY were used for GVHD prophylaxis.aGVHD grade !II and cGVHD (limited or extensive) were observed in four patients each before relapse.Eleven patients were diagnosed with hematological relapse, and seven were diagnosed with molecular or cytogenetic relapse.The median interval from HSCT to relapse was 3.0 (range, 0.6-27.3)months. In the case of non-haplo-HSCT, the median injection number was 2 (range, 1-4) (Supplementary material 3).The median total number of infused CD3 + T cells was 5.4×10 7 / kg (range, 0.1-25.3×10 7/kg).The median cell dose from the first to fourth DLI was 1.0, 3.0, 3.4 and 3.7×10 7 /kg, respectively.In the case of haplo-HSCT, the median initial cell dose was 0.05×10 7 /kg (range, 0.0001-0.1×10 7/kg).The median total cell dose was 0.73×10 7 /kg (range, 0.1-11.68×10 7/ kg).The median time from relapse to the onset of DLI was 1.5 (range, 0.3-36.8)months.The interval of DLI was not constant because of the retrospective nature of the study.Although the period between each injection was decided at the discretion of each attending physician, the timing of DLI de- pended on the changes in blast percentages, chimerisms or WT1 values in patients with hematological, cytogenetic or molecular relapse, respectively, as well as the occurrence of GVHD.Eventually, the median interval became 21 (range, 7-91) days, which was similar to that in previous studies (7,8). Concomitant treatment and post-DLI treatment Eight patients were treated with DLI together with Aza.The median initial dose of Aza was 53 mg/m 2 for 5-7 days.However, delayed administration was observed in four pa-tients due to hematotoxicity.The median number of Aza cycles was 8 (range, 4-20).Concomitant chemotherapies other than Aza were idarubicin/cytarabine (Ara-C) in two patients, enocitabine (BHAC)-based chemotherapies in two patients, mitoxantrone/etoposide/Ara-C (MEC) in one patient and Ara-C / aclarubicin / granulocyte colony-stimulating factor (CAG) in two patients.Following DLI, seven patients underwent allo-HSCT, including unrelated BM transplantation (U-BMT) in two patients, unrelated cord blood transplantation (U-CBT) in two patients and haploidentical peripheral blood stem cell transplantation (haplo-PBSCT) in three patients.Ten patients were treated with various chemotherapies, consisting of BHAC/daunorubicin, CAG, Ara-C, hydroxyurea, Mogamulizumab and gemtuzumab ozogamicin.All ALL patients were treated with DLI before 2018 when bispecific Tcell engager and antibody-drug conjugate (ADC) were available in Japan.Therefore, these patients were not treated with these newly introduced agents. GVHD after DLI and hematological toxicity aGVHD I-IV was observed in 14 (48.3%)patients following DLI (Table 2).The cumulative incidence of aGVHD of grade !II at 4 months after the initiation of DLI was 31.0%(Fig. 1A).No grade IV aGVHD was observed.Organs involved with aGVHD were the skin in 11 patients, colon in 6 patients and liver in 4 patients.Extensive cGVHD occurred in 3 (10.3%)patients, involving the liver in 1 patient and lung in 2 patients.In haplo-HSCT, the aGVHD grade !II incidence was 33.3%, while the incidence of extensive cGVHD was 16.7% (Table 2).Changes in the peripheral blood following DLI in patients who were treated with DLI alone are shown in Supplementary material 4. Reductions in the number of white blood cells, hemoglobin concentration and platelet count following DLI occurred in 6, 6 and 7 patients, respectively, out of 12 for whom data were available.We observed severe pancytopenia in one patient (patient #30).He was treated with G-CSF and transfusions of red blood cells and platelets, and his pancytopenia resolved after two weeks. Efficacy of DLI While 15 patients achieved CR, including 3 with hematological CR and 12 with molecular/cytogenetic CR, 14 showed progression of disease.Ten of the 15 patients who achieved CR maintained CR, while the other 5 with CR as their best response relapsed later.The overall response rate was 51.7% (Fig. 1B, Table 3).The CR rate in AML patients with hematological relapse was 33.3%.The CR rate was significantly higher in the patients with molecular relapse than in those with hematological relapse (81.8% vs. 27.3%,p=0.03).Similarly, a higher CR rate was observed in patients who developed aGVHD of grade !II or extensive cGVHD following DLI than in patients without GVHD.The median period to achieve CR from the initiation of DLI was 40 (range, days.The median duration of CR was 11.0 (range, 1.0-65.7)months.Cumulative relapse rates at 24 and 60 months following DLI in patients who achieved CR were 21.4% and 30.0%, respectively (Fig. 1C) Poor/adverse genetic risk group in AML, molecular relapse, longer interval from allo-HSCT to relapse and aGVHD of grade !II or extensive cGVHD following DLI were found to be significantly associated with a better response to DLI (Table 3).There was no significant correlation with the response rates among other factors analyzed, such as the diagnosis, allo-HSCT type, conditioning regimen, disease status at allo-HSCT, aGVHD or cGVHD before DLI, infused cell dose or concomitant chemotherapy (Table 3). The median OS from the initiation of DLI was 8.7 months (95% confidence interval, 5.5-29.8months).The OS rates at 1, 2 and 3 years after DLI were 41.4%, 37.9% and 30.3%, respectively (Fig. 2A).A log-rank analysis revealed that molecular/cytogenetic relapse, an interval from HSCT to relapse exceeding six months and concomitant chemotherapy with Aza were significantly associated with a relatively long survival following DLI (Table 4, Fig. 2B-D).The age, gender, diagnosis, disease status at HSCT, HSCT type, conditioning regimen, GVHD prophylaxis and presence of aGVHD or cGVHD before relapse were not associated with the survival.Factors related to DLI, such as the T cell dose, post-DLI therapy, response to DLI and GVHD following DLI did not significantly influence the survival (Table 4). Discussion The prognosis of the patients who relapsed after HSCT is poor, and therapeutic options are limited (2-4, 7), with no standard approach yet established.Previous studies have demonstrated that the efficacy of DLI is primarily limited to chronic-phase CML and indolent lymphoma (7,8).In the present study, we analyzed the efficacy of DLI in patients with acute leukemia or MDS who relapsed after allo-HSCT.We found that DLI was effective in approximately 50% of patients and that molecular or cytogenetic relapse, a relatively long interval from HSCT to relapse and concomitant chemotherapy with Aza were significantly associated with a relatively long survival following DLI. The CR rate in AML patients who had hematological relapse was 33.3%, which is fairly consistent with the CR rates reported by Shiobara (8) and Miyamoto (38% and 17%, respectively) (11).However, in the case of preemptive DLI, response rates based on the reduction of minimal residual disease (MRD) in molecularly relapsed acute leukemia have been reported to be 72% (26).In the present study, CR rates in patients with molecular or cytogenetic relapse were 81.8% and 42.9%, respectively, which appear to be consistent with those in previous reports (7,26). Regarding the survival, the 2-year survival rate of 37.9% in the present study seems to be consistent with those in previous reports regarding the 2-year OS in therapeutic DLI in AML, MDS and ALL (25-40%, 28-40% and 5-13%, respectively) (26-29).However, a direct comparison between our results and those reported previously is difficult due to differences in patient characteristics. We found that the development of aGVHD of grade !II following DLI was significantly associated with a high CR rate (Table 3), suggesting the involvement of a GVL effect mediated by the immune response against allo-HLA or minor histocompatibility antigens in close relation to GVHD.However, neither aGVHD nor cGVHD was associated with a relatively long survival after DLI (Table 4).It has been shown that high-grade GVHD tends to be complicated with severe infection and organ toxicity leading to death (5).In fact, the progression of GVHD and infection were the most common causes of death in patients who had aGVHD of grade !II or extensive cGVHD, while disease progression was the most common cause of death in patients who had no GVHD.Therefore, higher mortality rates due to complications related to GVHD may account for the decrease in the survival rate.The CR and 2-year survival rates were significantly higher in patients with molecular relapse than in those with hematological relapse in the present study (81.2% and 54.5%, respectively vs. 27.3% and 9.1%, respectively) (Table 3, 4).The results implied that the disease status at relapse strongly influenced the response to DLI and survival duration.Schmid et al. reported that the 2-year OS of therapeutic DLI was higher in patients who were treated in remission than in those with active disease or aplasia (56± 10% vs. 15±3%) (9).Several studies have demonstrated that the efficacy of DLI is superior in patients who are treated with DLI prophylactically or preemptively than in patients who are treated therapeutically (7,(12)(13)(14)27).These studies found that the 2-year OS rates in prophylactic or preemptive DLI for AML patients were 67-76% and 62-78%, respectively, while the 2-year OS in therapeutic DLI was only 7-25%.Therefore, these results indicate that a lower disease burden is associated with better outcomes and highlight the advantage of prophylactic or preemptive DLI over therapeutic DLI. Aza has been shown to augment immunity against malignancy by enhancing the expression of tumor-related antigens and HLA molecules and interferon responses (30).Reflecting the advantages of the anti-leukemic effects and immunomodulatory properties of Aza, there are several studies demonstrating a favorable outcome of the combination of Aza and DLI treatment for post-HSCT relapse in patients with AML or MDS (response rate: 30-37%; 2-year OS: 29-35%) (26, 31, 32).Consistent with previous studies, we found that DLI and concomitant treatment with Aza was significantly associated with a relatively long survival (2year OS: 75%), although the number of patients was small (Table 4). Haplo-HSCT has recently been introduced in clinics.There was some concern that GVHD following DLI might be more severe with haplo-HSCT than with matched related HSCT because of HLA mismatch.We found that the cumulative incidence rates of aGVHD II-IV and cGVHD were 33.3% and 16.7%, respectively, which were almost the same as in other types of HSCT (Table 2).There were no significant differences in the CR rates or 2-year OS between haplo-HSCT and other types of HSCT, which is consistent with previous studies (18-21).Therefore, DLI appears feasible and effective in haplo-HSCT using lower cell doses than other types of HSCT. A major limitation of this study is the fact that it was a retrospective one.Various factors regarding DLI and chemotherapies, such as the criteria for starting treatment, cell dose and interval of DLI and selection of a chemotherapy regimen thus varied among patients, which might have affected the clinical outcome.A prospective study with a sufficient number of patients is needed to draw a definitive conclusion, although it may be difficult to recruit a large number of eligible patients. In conclusion, while this study involved only a small number of patients and was a retrospective analysis, we found that DLI was beneficial for patients with acute leukemia or MDS who relapsed after allo-HSCT.The disease status at relapse was critical for better outcomes.Concomitant chemotherapy with Aza was significantly associated with a relatively long survival following DLI.These results suggest that DLI in combination with Aza for molecular or cytogenetic relapse may result in favorable outcomes.Precise monitoring of MRD using a molecular marker, such as WT1, is implied to be of great importance for this purpose. Figure 1 . Figure 1.Cumulative incidence of aGVHD and relapse rate evaluated by Gray's test following DLI and CR rates according to the diagnosis and hematological or molecular relapse.(A) Incidence of aGVHD.(B) CR rates.(C) Relapse rate. Figure 2 . Figure 2. Kaplan-Meier estimates of the OS of patients treated with DLI.(A) The OS of all patients.(B) The OS according to the disease status at relapse.(C) The OS according to the interval from allo-HSCT to relapse.(D) The OS according to concomitant treatment. Table 3 . Factors Associated with the Response Rates of DLI. ‡ Statistical significance of the differences among the groups was evaluated by Fisher's exact test.p<0.05 is considered significant and indicated in bold.Aza: 5-azacytidine Other abbreviations are the same as listed in Table2.	2023-05-25T15:20:23.221Z	2023-05-24T00:00:00.000	{ "year": 2023, "sha1": "f4fbad54cd7866c3493b7037994020016c7638d1", "oa_license": "CCBYNCND", "oa_url": "https://www.jstage.jst.go.jp/article/internalmedicine/advpub/0/advpub_1714-23/_pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "f2ccf4f2bede088c79371dfd22880f324b33a411", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
13145922	pes2o/s2orc	v3-fos-license	Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α* Background: Extracellular Ca2+ alters mGluR1α activity but by an unknown mechanism. Results: Mutations in predicted Ca2+-binding sites modulated the potency of both orthosteric and allosteric modulators. Conclusion: Ca2+ binding exerts multiple types of effects on mGluR1α. Significance: Improved knowledge of the mechanisms underlying the actions of Ca2+ on mGluR1α activity could facilitate development of isoform-selective drugs and/or suggest ways to tune the actions of available drugs. Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. Metabotropic glutamate receptor 1␣ (mGluR1␣), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1␣ is also modulated by extracellular Ca 2؉ . However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca 2؉ -binding site(s) on mGluR1␣ may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca 2؉ -binding site in the hinge region of mGluR1␣ is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca 2؉ enhanced mGluR1␣-mediated intracellular Ca 2؉ responses evoked by the orthosteric agonist L-quisqualate. Conversely, extracellular Ca 2؉ diminished the inhibitory effect of the mGluR1␣ orthosteric antagonist (S)-␣-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1acarboxylate ethyl ester) allosteric modulators of mGluR1␣ potentiated and inhibited responses to extracellular Ca 2؉ , respectively, in a manner similar to their effects on the response of mGluR1␣ to glutamate. Mutations at residues predicted to be involved in Ca 2؉ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist L-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca 2؉ . These studies reveal that binding of extracellular Ca 2؉ to the predicted Ca 2؉binding site in the extracellular domain of mGluR1␣ modu-lates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1␣. The eight subtypes of metabotropic glutamate receptors (mGluRs) 2 belong to family C of the G protein-coupled receptors (GPCRs) and possess a large extracellular domain (ECD), a transmembrane domain (TMD), and a cytosolic C-terminal tail. The mGluRs are widely expressed in the central nervous system and play critical roles in regulating neuronal excitability and synaptic plasticity at both excitatory and inhibitory synapses (1). Extensive structural studies have revealed that the endogenous agonist L-glutamate (L-Glu), the major excitatory neurotransmitter in the central nervous system, binds at the hinge region of the ECD within the Venus fly trap motif of the receptor to activate the protein. This subsequently stimulates phospholipase C and leads to accumulation of inositol trisphosphate and an increase of intracellular calcium concentration ([Ca 2ϩ ] i ) (2)(3)(4). In recent years, mGluRs have received increasing interest as potential drug targets for the treatment of a range of psychiatric and neurological diseases (5) (see Fig. 1). The ligands targeting mGluRs can be classified as orthosteric agonists and antagonists as well as allosteric modulators. Orthosteric agonists and antagonists induce and attenuate, respectively, the activity of the receptor by competitively binding to the L-Glu-binding pocket. L-Quisqualate (L-Quis), the most potent agonist of mGluR1 reported to date (6,7), has been speculated to share nearly the same binding pocket as L-Glu (8,9). In contrast, (S)-MCPG is an analog of L-Glu and is a non-selective competitive antagonist that has been shown to occupy the L-Glu-binding pocket, thereby blocking the function of group I/II members in the mGluR family (10). On the other hand, allosteric modulators bind to sites other than the orthosteric center to affect the activity of the receptor. Ro 67-4853 is a positive allosteric modulator (PAM) of mGluR1 that enhances the potency of L-Glu by interacting with the TMD of the receptor. CPCCOEt is a negative allosteric modulator (NAM) that inhibits the activation of mGluR1 by L-Glu by specifically binding to a site that involves the third extracellular loop of mGluR1␣ (11). Like other members of the family C GPCRs, such as the calcium-sensing receptor, mGluR1␣ senses [Ca 2ϩ ] o using the extracellular domain (12,13). By transient expression of mGluR1␣ in oocytes, Kubo et al. (4) demonstrated that mGluR1-mediated activation of Ca 2ϩ -activated Cl Ϫ channels is modulated by [Ca 2ϩ ] o in addition to L-Glu. Purkinje cells from mGluR1 knock-out mice lose sensitivity to [Ca 2ϩ ] o , and this sensitivity to [Ca 2ϩ ] o was restored after mGluR1 was genetically reintroduced into the mice (14). There are sparse reports of [Ca 2ϩ ] o affecting the action of various classes of compounds acting on mGluRs (15). However, it is not clear how [Ca 2ϩ ] o is able to modulate the activity of mGluR1 or the actions of various mGluR1 ligands, and no Ca 2ϩ -binding sites have been identified in the 15 structures solved by x-ray crystallography to date (Protein Data Bank). Using our recently developed computational algorithm, we identified a novel potential [Ca 2ϩ ] o -binding site within the hinge region of the ECD of mGluR1␣ adjacent to the reported L-Glu-binding site (16,17). It comprises Asp-318, Glu-325, Asp-322, and the carboxylate side chain of the natural agonist L-Glu. The carboxylate side chains of both L-Glu and Asp-318 are involved in both L-Glu and [Ca 2ϩ ] o binding. Our previous mutagenesis study indicated that binding of L-Glu and Ca 2ϩ to their distinct but partially overlapping binding sites synergistically modulates mGluR1␣-mediated activation of [Ca 2ϩ ] i signaling. Mutating the L-Glu-binding site completely abolished L-Glu signaling but left its Ca 2ϩ -sensing capability largely intact. Mutating predicted Ca 2ϩ -binding residues not only abolished or significantly reduced the sensitivity of mGluR1␣ to [Ca 2ϩ ] o but also in some cases to L-Glu (18). In the present study, we first demonstrated that our predicted Ca 2ϩ -binding site is adjacent to the orthosteric agonist and antagonist interaction sites. We then examined the role of [Ca 2ϩ ] o in modulating the actions of different orthosteric ligands acting on mGluR1␣, including L-Quis and (S)-MCPG as well as reciprocal interactions between Ca 2ϩ and the mGluR1 allosteric modulators Ro 67-4853 and CPCCOEt. Our results suggest that [Ca 2ϩ ] o modulates the sensitivity of mGluR1␣ to not only orthosteric agonists and antagonists but also to allosteric modulators likely by interacting with the predicted [Ca 2ϩ ] o -binding site in the ECD of the receptor. EXPERIMENTAL PROCEDURES Docking L-Quis to ECD-mGluR1␣ Using AutoDock Vina and Hinge Motion Analysis-To elucidate binding of L-Quis to the ECD of mGluR1␣, L-Quis was docked into the crystal structure (Protein Data Bank 1EWK). After removing the coordinates of the bound endogenous ligand, L-Glu, the Protein Data Bank file was loaded into AutoDock tools to add polar hydrogen atoms and choose the docking center and grid box. The docking work was carried out by the AutoDock tool Vina (Scripps). The binding residues were analyzed by measuring the atoms within 6 Å of L-Quis. The L-Glu-and the (S)-MCPG-binding sites within the hinge region were analyzed using Dymdon. Molecular Dynamics Simulation and Correlation Analysis Using AMBER-The initial coordinates for all the simulations were taken from a 2.20-Å resolution x-ray crystal structure (Protein Data Bank code 1EWK; Ref. 19). The AMBER 10 suite of programs (20) was used to carry out all of the simulations in an explicit TIP3P (transferable intermolecular potential 3P) water model (21) using the modified version of the all-atom Cornell et al. (22) force field and the reoptimized dihedral parameters for the peptide -bond (23). The crystal structure contains only Glu substrate. Ca 2ϩ ion was placed at the suggested Ca 2ϩ -binding site that is defined by residues Asp-318, Asp-322, and Glu-325. An initial 2-ns simulation was performed using NOE restraint during the equilibration to reorient the side chain residues in the Ca 2ϩ -binding site, but no restraints were used during the actual simulation. A total of four molecular dynamics simulations were carried out for 50 ns each on wild type and three mutant mGluRs. The mutations were D318I, D322I, and E325I. First, our structures were minimized to achieve the lowest energy conformation in each complex. The structures were then equilibrated for 2 ns, starting the molecular dynamics simulations from the equilibrated structures. During the simulations, an integration time step of 0.002 ps was used to solve Newton's equation of motion. The long range electrostatic interactions were calculated using the particle mesh Ewald method (24), and a cutoff of 9.0 Å was applied for non-bonded interactions. All bonds involving hydrogen atoms were restrained using the SHAKE algorithm (25). The simulations were carried out at a temperature of 300 K and a pressure of 1 bar. A Langevin thermostat was used to regulate the temperature with a collision frequency of 1.0 ps Ϫ1 . The trajectories were saved every 500 steps (1 ps). The trajectories were then analyzed using the ptraj module in AMBER 10. Constructs, Site-directed Mutagenesis, and Expression of mGluR1␣ Variants-The red fluorescent protein mCherry was genetically tagged to the C terminus of mGluR1␣ by a flexible linker, GGNSGG (18). Point mutations were introduced using a site-directed mutagenesis kit (Stratagene). HEK293 cells were seeded and cultured on glass coverslips. mGluR1␣ and its mutants were transfected into cells utilizing Lipofectamine 2000 (Invitrogen). The cells were then incubated for an additional 2 days so that mGluR1␣ and its mutants were expressed at sufficient levels for study. Cells were fixed on the coverslips with 4% formaldehyde, and nuclei were stained with DAPI. The expression of mGluR1␣ and its variants was detected by measuring red fluorescence using confocal microscopy at 587 nm. Determining the Effect of [Ca 2ϩ ] o on Activation of mGluR1␣ and Its Mutants by L-Quis-Measurement of [Ca 2ϩ ] i was performed as described (13). In brief, wild type mGluR1␣ was transiently transfected into the cells and cultured for an additional 2 days. The cells on the coverslips were subsequently loaded using 4 M Fura-2 AM in 2 ml of physiological saline buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 0.55 mM MgCl 2 , and 1 mM CaCl 2 , pH 7.4) for 30 min. The coverslips were then mounted in a bathing chamber on the stage of a fluorescence microscope at room temperature. Fura-2 emission signals at 510 nm from single cells excited at 340 or 380 nm were collected utilizing a Leica DM6000 fluorescence microscope in real time as the concentration of L-Quis was progressively increased in the presence or absence of [Ca 2ϩ ] o . The ratio of fluorescence emitted at 510 nm resulting from excitation at 340 or 380 nm was further analyzed to obtain the [Ca 2ϩ ] i response as a function of changes in L-Quis. Only the individual cells expressing mCherry were selected for analysis. Determining the Effects of [Ca 2ϩ ] o on the Potency of Ro 67-4853 on mGluR1␣-Fura-2 AM was used for monitoring [Ca 2ϩ ] i in real time as described above. Ro 67-4853 did not potentiate mGluR1␣ in the absence of L-Glu (26,27). To obtain the [Ca 2ϩ ] i readout, HEK293 cells expressing mGluR1␣ were preincubated with 0.5 mM Ca 2ϩ and 5 nM Ro 67-4853 for at least 10 min. Cells loaded with Fura-2 AM were mounted onto a chamber perfused with saline buffer. The concentration of Ro 67-4853 was increased stepwise in the presence of 0. 5 Binding to mGluR1␣ and Its Mutants-HEK293 cells transiently transfected with wild type mGluR1␣ or its mutants were maintained in a 5% CO 2 37°C incubator for an additional 48 h as before. Cells were then collected in ice-cold hypotonic buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 5 mM KCl, 0.5 mM EDTA, and 1% protease inhibitors at pH 7.0 -7.5). The cell pellet was washed twice more using hypotonic buffer to remove the L-Glu in the cellular debris. The crude membrane protein (100 g) was mixed with 30 nM L-[ 3 H]Quis in 100 l of hypotonic buffer. The nonspecific binding was determined by measuring bound Predicted [Ca 2ϩ ] o -binding Site Is Adjacent to Orthosteric Agonist and Antagonist-binding Sites-Using our recently developed computational algorithms, we have identified a novel potential Ca 2ϩ -binding site at the hinge region of the ECD of mGluR1␣ (18). Fig. 1 shows that the predicted Ca 2ϩbinding site comprises Asp-318, Glu-325, Asp-322, and the carboxylate side chain of the natural agonist L-Glu in the hinge region in the ECD of mGluR1␣ adjacent to the reported L-Glubinding site. Asp-318 is involved in both L-Glu and Ca 2ϩ binding (18). Using the crystal structure (Protein Data Bank code 1EWK; closed-open form) of the ECD of the receptor and the AutoDock Vina program, we modeled the binding site for the orthosteric agonist L-Quis. As shown in Fig. 1B, the docked binding site of the agonist L-Quis corresponds well with the L-Glu-binding residues previously suggested by the crystal structure. Our predicted Ca 2ϩ -binding site is also adjacent to the L-Quis pocket and interacts with L-Quis similarly to L-Glu (Fig. 1B). In the reported crystal structure of mGluR1 complexed with an orthosteric antagonist, (S)-MCPG (Protein Data Bank code 1ISS), (S)-MCPG interacts with Tyr-74, Trp-110, Ser-165, Thr-188, and Lys-409 in lobe 1 and Asp-208, Tyr-236, and Asp-318 in lobe 2 ( Fig. 1B) (10). It shares with L-Glu most of the residues of the L-Glu-binding pocket (10) and is also adjacent to our predicted Ca 2ϩ -binding site. We next performed molecular dynamics simulations to reveal any possible interaction between our predicted [Ca 2ϩ ] obinding site and the orthosteric ligand-binding site. Residues involved in the [Ca 2ϩ ] o -binding pocket, such as Asp-318, Asp-322, and Glu-325, have strong correlated motions as expected given their roles as [Ca 2ϩ ] o -binding ligands. In addition, residues Asp-318 and Arg-323 residing within the same loop as the predicted Ca 2ϩ -binding site are also concurrently correlated. As shown in Fig. 2, most of the critical L-Glu-binding residues, including Trp-110, Ser-165, Thr-188, Asp-208, Tyr-236, Asp-318, and Arg-323, are well correlated to the [Ca 2ϩ ] o -binding site (Asp-318, Asp-322, and Glu-325). However, mutations at the charged residues involved in [Ca 2ϩ ] o binding, such as D318I and E325I, markedly attenuated the correlation of the Ca 2ϩbinding site with the L-Glu-binding pocket. The Ca 2ϩ -binding site in mutant D318I only correlates with Gly-293 and Asp-208, and mutant D325I only correlates with Tyr-236 and Gly-293. The mutant D322I also exhibited impaired correlation between the [Ca 2ϩ ] o -binding site and L-Glu-binding site but to a lesser degree. As shown in Table 1, Asp-318 in the [Ca 2ϩ ] o -binding site still correlates with four residues in the L-Glu-binding pocket (Fig. 2). Similarly, residues that are involved in binding L-Quis and (S)-MCPG also correlate well with residues involved in the predicted [Ca 2ϩ ] o -binding site. Results from these analyses and our previous studies on the effect of binding of [Ca 2ϩ ] o to its site on L-Glu-mediated activation of mGluR1 led us to hypothesize that [Ca 2ϩ ] o regulates the effects of orthosteric ligands on mGluR1␣. Ca 2ϩ Enhances Sensitivity of Activation of mGluR1␣ by L-Quis by Increasing L-[ 3 H]Quis Binding via Interaction with the [Ca 2ϩ ] o -binding Site of the Receptor-To test the effect of [Ca 2ϩ ] o on the activation of mGluR1␣ by the orthosteric agonist L-Quis, we performed a single cell fluorescence imaging assay by measuring changes in [Ca 2ϩ ] i using HEK293 cells tran-siently transfected with mGluR1␣ and loaded with Fura-2. To eliminate any potential effect of trace L-Glu secreted from cells, experiments were conducted using continuous superfusion of cells with an L-Glu-free buffer. Fig. 3, A-D, show that L-Quis induced intracellular calcium responses mediated by mGluR1 in a manner similar to the activation of the receptor by L-Glu. [Ca 2ϩ ] o behaved as a PAM of the L-Quis response and induced a leftward shift in the L-Quis concentration-response curve for activation of mGluR1a (Fig. 3, A-D). In the absence of [Ca 2ϩ ] o (Ca 2ϩ -free buffer with less than 2 M calcium), the EC 50 for the activation of mGluR1a by L-Quis is 12.8 nM. The addition of 1.8 Table 2). Importantly, this mutation significantly reduced the [Ca 2ϩ ] o -mediated enhancement in potency for L-Quis from 4.6-to 1.6-fold in 1.8 mM [Ca 2ϩ ] o , although both the potency and efficacy of L-Quis-mediated activation of the E325I mutant were still enhanced rela-tive to WT mGluR1 (Fig. 3, A-D). As L-Glu could potentially serve as a ligand for binding of Ca 2ϩ to its pocket, L-Glu or L-Quis binding could rescue the mutated Ca 2ϩ -binding pocket, thus enhancing the Ca 2ϩ sensitivity of the mutant. On the other hand, mutant D322I exhibited WT-like behavior in its response to L-Quis both in the absence and presence of [Ca 2ϩ ] o (Fig. 3, A-D, and Table 2), consistent with Asp-322 contributing to [Ca 2ϩ ] o binding to a lesser degree with only its main chain oxygen serving as a ligand atom. We also observed WT-like modulation of the L-Glu response of D332I by Ca 2ϩ (18). These Table 4). The maximal response was also significantly decreased by 40 M CPCCOEt, although the maximal response with 5 M CPCCOEt was still comparable. This indicates that 30 mM [Ca 2ϩ ] o cannot completely reverse the antagonism induced by CPCCOEt, and thus the inhibitory effects of CPCCOEt on the response of mGluR1␣ to [Ca 2ϩ ] o appear to be non-competitive ( Fig. 5B and Table 4). The mGluR1␣ PAM Ro 67-4853 Potentiates Activation of mGluR1 by [Ca 2ϩ ] o -The finding that CPCCOEt inhibited activation of mGluR1 by [Ca 2ϩ ] o suggests that the CPCCOEt site in the transmembrane-spanning domain of mGluR1 and the [Ca 2ϩ ] o -binding site in the ECD of the receptor interact in a manner similar to the interactions between the orthosteric L-Glu-binding site and the allosteric CPCCOEt site. We performed analogous experiments to determine whether the mGluR1 PAM Ro 67-4853, which binds to the extracellular loops of the TMDs of mGluR1␣ (2, 29) (Fig. 1B), can also potentiate responses to [Ca 2ϩ ] o . Fig. 6A shows that L-Glu-induced activation of WT mGluR1␣ was enhanced by the addition of 10 or 100 nM Ro 67-4853 using single cell [Ca 2ϩ ] i imaging. We then examined the effects of Ro 67-4853 on the [Ca 2ϩ ] o sensitivity of wild type mGluR1␣ in the absence of L-Glu. Fig. 6B shows that both 30 TABLE 3 Addition of 0.5 mM (S)-MCPG decreases the responses of mGluR1␣ to [Ca 2؉ ] o and L-Glu The [Ca 2ϩ ] i response to [Ca 2ϩ ] o and L-Glu in the absence or presence of 0.5 mM (S)-MCPG were obtained by measuring the ratiometric change of Fura-2 AM fluorescence. Response to [Ca 2؉ ] o Response to L-Glu 6B and Table 5). To further evaluate the effect of Ro 67-4853 on mGluR1␣, HEK293 cells transiently expressing mGluR1␣ were preincubated with 0.5 mM Ca 2ϩ and 5 nM Ro 67-4853 for up to 10 min, and then the responses to multiple concentrations of Ro 67-4853 were tested. In the presence of 0.5 mM [Ca 2ϩ ] o , Ro 67-4853 enhanced L-Glu-induced mGluR1␣ activity in a concentration-dependent manner. Increasing [Ca 2ϩ ] o to 1.8 mM significantly increased the potency of a low dosage of Ro 67-4853 for mGluR1␣ (p Ͻ 0.05) (Fig. 6C). At the same time, the EC 50 value decreased from 20.7 to 10.0 nM (Fig. 6C and Table 5). Interestingly, [Ca 2ϩ ] i oscillations were observed when the cells were treated with Ro 67-4853 (data not shown). Similar to the Ca 2ϩ -sensing receptor, three different patterns of response were noted (30) Fig. 1), we then performed studies using an mGluR variant with a key [Ca 2ϩ ] o -binding ligand residue mutated, E325I. Fig. 1B shows that Glu-325 is not directly involved in L-Glu binding, and variant E325I is able to sense L-Glu in a manner similar to WT (18). Fig. 7A shows that addition of 30 M L-Glu enhanced the responsiveness of E325I to Ro 67-4853. Of note, Fig. 7B shows that E325I responded to 10 M Ro 67-4853 in the absence of L-Glu in [Ca 2ϩ ] o -free saline. Increasing [Ca 2ϩ ] o from 0.5 to 1.8 mM did not affect the sensitivity of E325I to Ro 67-4853, but elevating [Ca 2ϩ ] o increased the responses of WT mGluR1␣ to 300 nM Ro 67-4853 (Fig. 7B). This suggests that mutating the Ca 2ϩ -binding site (E325I) eliminates the effect of Ca 2ϩ on Ro 67-4853 but not on WT mGluR1␣. To determine whether the receptors were saturated by Ro 67-4853, higher concentrations of the PAM were applied to both WT mGluR1 and E325I. As shown in Fig. 7B, higher concentrations of Ro 67-4853 increased the responses of both WT mGluR1 and E325I. This result suggests that [Ca 2ϩ ] o binding at its predicted site in the hinge region is essential for the positive allosteric action of this modulator. DISCUSSION In this study, we demonstrated that [Ca 2ϩ ] o had significant modulating effects on the actions of various orthosteric and allosteric ligands on mGuR1a as assessed using a functional readout (i.e. [Ca 2ϩ ] i responses) in receptor-transfected HEK293 cells. [Ca 2ϩ ] o exerted several different effects on the compounds studied here, including the orthosteric agonist L-Quis, the orthosteric antagonist (S)-MCPG, and allosteric modulators, e.g. the PAM Ro 67-4853 and the NAM CPCCOEt. As shown in Fig. 1, the predicted [Ca 2ϩ ] o -binding site partially overlaps the predicted orthosteric binding site for the agonist L-Quis and the antagonist (S)-MCPG. We have previously (18). However, activation of GPCRs is also known to induce Ca 2ϩ influx through store-operated Ca 2ϩ entry channels (31,32). By utilizing Gd 3ϩ , an inhibitor of these Ca 2ϩ channels, we noted that mGluR1a still could induce an increase in [Ca 2ϩ ] i (18 (Fig. 3). ] o -binding site with the adjacent binding site for orthosteric agonists and antagonists. We first showed that the L-Quis-binding pocket predicted here using AutoDock Vina overlaps extensively with the L-Glu-binding pocket in the reported crystal structure ( Table 6). The side chain of Asp-318 is involved in both [Ca 2ϩ ] o and agonist binding. In our earlier study, the (18). In this study, it also completely eliminated L-Quis-mediated activation of mGluR1 (Fig. 3E). This finding is supported by a previous report that the mutants T188A, D208A, Y236A, and D318A abolished the sensitivity of the receptor to both L-Quis and L-Glu, whereas the mutants R78E and R78L exhibited clearly impaired L-Quis binding (8,9). The key residue Glu-325 is involved in [Ca 2ϩ ] o binding, and the mutant E325I indeed significantly impaired both the [Ca 2ϩ ] o and L-Glu sensitivity of the receptor (Fig. 3). On the other hand, variant D322I produced less reduction of the modulatory effects of [Ca 2ϩ ] o on both L-Quis and L-Glu agonist action, which is consistent with its lesser role in [Ca 2ϩ ] o binding with a contribution of only a main chain ligand atom (Fig. 1). Our observed effect of [Ca 2ϩ ] o on responses to orthosteric agonists and antagonists of mGluR1 is consistent with molecular dynamics simulation studies performed here on the correlated motions of the hinge region in the ECD of mGluR ( Fig. 2 and Table 1). We observed a strong correlation among residues in the predicted [Ca 2ϩ ] o -binding site and residues involved in the orthosteric binding sites shared by L-Glu, L-Quis, and (S)-MCPG. Interestingly, mutation of the [Ca 2ϩ ] o -binding site largely removed this correlation. Fig. 1A shows that the predicted [Ca 2ϩ ] o -binding site at the hinge region is conserved in the group I mGluRs, e.g. mGluR1 and mGluR5 (18) (12,13,38,39). In recent years, increasing numbers of family C GPCRs have been found to exhibit synergistic modulation of the primary orthosteric agonist by allosteric modulators. Sweet enhancers binding to the hinge region of the human taste receptor are known to stabilize the active form of the receptor, thus leading to altered perception of sweet taste, whereas IMP and L-Glu also synergistically activate the umami taste receptor (40,41). It is also interesting to note that an allosteric ligand suggested to act at the ECD domain of mGluR is located at the hinge region (42,43). Thus, our work has strong implications for the role of the hinge region of the ECD in modulating action of small molecule ligands on family C GPCRs. As for allosteric modulators targeting the TMDs, the binding sites of positive and negative modulators of mGluR1␣ are distinct (44). These allosteric modulators effectively modulate receptor activation by L-Glu, but little is known about the effects of the endogenous mineral ion Ca 2ϩ on these modulators. In this study, the effects of [Ca 2ϩ ] o on CPCCOEt (NAM) and Ro 67-4853 (PAM) were further assessed. The non-competitive NAM CPCCOEt is known to inhibit the L-Glu response by binding to Thr-815 and Ala-818 on the seventh transmembrane helix (45,46). Our data shown in Fig. 5 support the contention that CPCCOEt, acting as a non-competitive inhibitor, also can diminish the [Ca 2ϩ ] i response of mGluR1␣. Interestingly, increasing [Ca 2ϩ ] o restored the [Ca 2ϩ ] o sensitivity of the receptor. CPCCOEt not only inhibits proliferation of melanoma cells but also reverses morphine tolerance (47,48). Thus, the findings in this study indicate that a novel drug targeting the [Ca 2ϩ ] o -binding site in mGluR1 has the potential to tune the therapeutic effect of CPCCOEt on melanoma or addiction. Val-757 in the TMD was revealed to be critical to the activation of mGluR1 by the PAMs (27,44 (Fig. 7). PAMs binding to the TMDs have been shown to enhance L-Quis binding to mGluR1␣ (27). It is possible that the incomplete reduction in the inhibitory effect of MCPG by [Ca 2ϩ ] o is due to an additional synergistic effect involving the TMD region of the receptor. By tagging the FRET pair YFP/cyan fluorescent protein to the two intracellular loops 2 (i2) of the dimeric mGluR1␣, Tateyama et al. (49) observed that the rearrangement of the TMD induced by L-Glu was reversed by (S)-MCPG. Such an integrated effect of the TMD with the ECD region is further supported by studies of mGluRs with deletions of the Venus fly trap. It was found that PAMs not only potentiate the action of agonists on the full-length receptors but sometimes can display strong agonist activity on Venus fly trap-truncated receptors (50,51). The Venus fly traps of the ECDs are not only responsible for agonist-induced activation but also prevent PAMs from activating the full-length receptor (50,51). Taken together, our study reveals that [Ca 2ϩ ] o binding at the hinge region is likely to be responsible for its capacity to modulate action of other allosteric modulators. Tables 4 and 5). Over the past decade, many new PAMs and NAMs for various receptors have been developed, and the potential exists for developing allosteric modulators with greater subtype specificity than is possible for orthosteric agonists (52). The co-activation induced by endogenous agonists and PAMs binding to the hinge regions of receptors could be a common feature of family C GPCRs. These data provide further insight into the modulation of mGluR1␣ by [Ca 2ϩ ] o and suggest that [Ca 2ϩ ] o has the potential to modulate the profile of a variety of agents acting on mGluR1␣, including agonists, antagonists, and allosteric modulators. In conclusion, we investigated the effects of [Ca 2ϩ ] o on the modulation of mGluR1␣ by orthosteric agonists and an orthosteric antagonist as well as by a PAM and NAM and found that [Ca 2ϩ ] o enhanced the actions of agonists and PAMs but attenuated the actions of antagonists and NAMs. These findings provide new insights into the targeting of mGluR1␣ by different classes of ligands. In addition to the specific relevance of these findings for understanding the nature of allosteric modulation of mGluR1␣, they may also have general relevance for understanding the modulation of family C GPCRs by extracellular ions, such as Ca 2ϩ .	2018-04-03T05:58:44.700Z	2013-11-26T00:00:00.000	{ "year": 2013, "sha1": "0c539f26c722af07cb8e4924080fa844136e4a7d", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/289/3/1649.full.pdf", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "3b5d0c1eae0cf57887a33647f6dc2429c7a404a5", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
30954081	pes2o/s2orc	v3-fos-license	Heparin Enhances Serpin Inhibition of the Cysteine Protease Cathepsin L* The glycosaminoglycan heparin is known to possess antimetastatic activity in experimental models and preclinical studies, but there is still uncertainty over its mechanism of action in this respect. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III, but a similar cofactor role has not been previously investigated for proteases linked to metastasis. The squamous cell carcinoma antigens (serpins B3 and B4) are tumor-associated proteins that can inhibit papain-like cysteine proteases, including cathepsins L, K, and S. In this study, we show that SCCA-1 (B3) and SCCA-2 (B4) can bind heparin as demonstrated by affinity chromatography, native PAGE gel shifts, and intrinsic fluorescence quenching. Binding was specific for heparin and heparan sulfate but not other glycosaminoglycans. The presence of heparin accelerated inhibition of cathepsin L by both serpins, and in the case of SCCA-1, heparin increased the second order inhibition rate constant from 5.4 × 105 to >108, indicating a rate enhancement of at least 180-fold. A templating mechanism was shown, consistent with ternary complex formation. Furthermore, SCCA-1 inhibition of cathepsin L-like proteolytic activity secreted from breast and melanoma cancer cell lines was significantly enhanced by heparin. This is the first example of glycosaminoglycan enhancement of B-clade serpin activity and the first report of heparin acting as a cofactor in serpin cross-class inhibition of cysteine proteases. Most importantly, this finding raises the possibility that the anticancer properties of heparin may be due, at least partly, to enhanced inhibition of prometastatic proteases. Serpin B3 was originally isolated as squamous cell carcinoma antigen (SCCA), 2 a tumor marker antigen associated with cervical cancer that has served as a diagnostic serum marker for squamous cell carcinomas of the cervix, head, neck, and lung (1,2). SCCA is an atypical serpin in that it exhibits cross-class activity, inhibiting papain-like cysteine proteases (cathepsins L, K, and S) rather than serine proteases as targeted by most family members (3,4). A closely related human gene encoding serpin B4 (SCCA-2) was subsequently isolated (5,6) and has 92% protein sequence identity to the original antigen (SCCA-1), with the most significant divergence in the reactive center loop region. SCCA-2 shows some overlap in inhibitory profile with SCCA-1, but it can inhibit the serine proteases cathepsin G and mast cell chymase and shows significantly weaker inhibition of cysteine proteases in comparison with SCCA-1 (7). In addition, SCCA-1 can inhibit parasite-derived cysteine proteases (8), and SCCA-2 inhibits the Der p 1 mite allergen cysteine protease activity (9). Overexpression of SCCA-1 in PCI-51 cells and keratinocytes has been shown to block tumor necrosis factor-␣and UV light-induced apoptosis, respectively (10,11), and SCCA-2 overexpression can protect HeLa cells from tumor necrosis factor-␣-induced apoptosis (12). It has been proposed by Silverman et al. (13) that, along with other human B-clade members, the major function of these serpins may be to protect cells against promiscuous proteolysis. However, the implied prosurvival role in cancer has not been clearly established, and in contrast, SCCA-1 overexpression in head and neck squamous carcinoma cells significantly inhibits in vitro migration in Matrigel assays and in vivo growth and tumor invasion in nude mice (14). Similarly, the depletion of SCCA-1 using an antisense approach results in increased invasive activity of SiHa cervical carcinoma cells (15). SCCA-1 and SCCA-2 lack a recognizable secretory signal sequence, and they appear to be predominantly cytoplasmic in the normal epithelium (16). However, the antigens are routinely found extracellularly in the plasma of patients with various malignant and nonmalignant diseases (17). In addition, SCCA-1 can be secreted upon treatment of keratinocytes and HEK293 cells with interleukin-4 and interleukin -13, and this appears to be independent of the endoplasmic reticulum/Golgi pathway (8). This may implicate the immune system in triggering SCCA-1 secretion in the allergic response and perhaps also during malignancy. Extracellular mammalian targets have not yet been characterized, but it is known that cysteine cathepsins are also up-regulated in many cancers, including malignant melanoma, where secreted cathepsins contribute to the breakdown of the extracellular matrix during metastasis (18,19). Serpin inhibition of proteases proceeds via a well characterized conformational switch mechanism, resulting in a stable complex containing a distorted inactivated protease (20). For a number of serpins, this activity can be enhanced by the presence of cofactors, most notably the effects of glycosaminoglycans on plasma serpin inhibition. Other ligands can also modulate serpin activity, and Ong et al. (21) showed that SCCA-1 activity against cathepsin V can be enhanced in the presence of DNA, but that unlike the nuclear serpin MENT, SCCA-1 does not bind DNA directly, and enhancement appears to be mediated via the protease. In this study, we initially investigated the possibility that SCCA-1 and SCCA-2 could bind glycosaminoglycans despite the fact that they possess an overall negative isoelectic point. Using a combination of solid phase and solution phase techniques, we found that these serpins bind heparin and heparan sulfate but not other glycosaminoglycans. Kinetic analysis showed that heparin significantly enhanced inhibition of the cysteine protease cathepsin L but had no effect on SCCA-2 inhibition of the serine protease cathepsin G. We also found that proteolysis of a cathepsin L substrate by extracellular fractions from MDA-MB-231 and WM793 cancer cell lines was more potently inhibited by SCCA-1 when in the presence of heparin. EXPERIMENTAL PROCEDURES Recombinant Protein Production-The full-length open reading frame cDNAs for SCCA-1, SCCA-2, and ovalbumin were previously cloned into the pRSETC expression vector (22). Escherichia coli BL21(DE3) cells transformed with plasmid were grown in 50 ml of Overnight Express autoinducing medium (Merck) containing 100 g/ml ampicillin for 16 h at 37°C, and this was used to inoculate 0.5-1 liters of Overnight Express medium. Following growth at 37°C for 24 h, cells were harvested by centrifugation at 11,000 rpm for 30 min and lysed using BugBuster lysis reagent (Merck). Soluble material was clarified by centrifugation of the cell lysate at 15,000 rpm for 30 min at 4°C, followed by 0.22 m filtration. The recombinant serpin was purified from this extract using a His⅐Bind purification kit (Merck), routinely yielding Ͼ5 mg of protein from 500 ml of culture. Binding to Heparin by Affinity Chromatography-Heparin HiTrap 1-ml columns (GE Healthcare) were equilibrated with buffer A (50 mM Tris and 20 mM NaCl, pH 6.9). 0.5 mg of recombinant SCCA (rSCCA)-1, rSCCA-2, recombinant ovalbumin, and antithrombin III in buffer A were applied, followed by 10 column volumes of buffer A. Bound proteins were eluted using a stepwise NaCl gradient (0 -1 M) in buffer A at 1.5 column volumes/fraction. Glycosaminoglycan Specificity-20 g of rSCCA-1 or rSCCA-2 was added to 50 l of a 50% slurry of heparin-agarose beads in buffer A containing 100 mM NaCl in a final volume of 200 l, followed by incubation at 4°C for 1 h. The beads were washed three times with buffer A to remove unbound protein. Heparin, heparan sulfate, acetyl heparin, de-N-sulfated heparin, or chondroitin sulfate A or B (all from Sigma) in buffer A (50 or 500 g/ml) was added to the beads and incubated for 10 min at 4°C. Beads were pelleted by centrifugation at 1000 rpm for 5 min, and supernatants were analyzed by SDS-PAGE. Intrinsic Tryptophan Fluorescence-The tryptophan fluorescence of rSCCA-1 and rSCCA-2 was monitored in both the presence and absence of heparin to investigate if heparin binding induces a conformational change. 1 M rSCCA was incubated with 1, 2, and 5 M heparin in cathepsin assay buffer (200 mM sodium acetate, 8 mM dithiothreitol, 4 mM EDTA, and 0.1% Brij-35, pH 5.5) in a final volume of 600 l. Using a Hitachi F4500 fluorometer, each sample was excited at 295 nm, and the emission was scanned over a wavelength range of 320 -400 nm at a rate of 60 nm/min using excitation/emission slit widths of 10 nm. The buffer fluorescence spectra (with or without heparin) were subtracted from each sample. Each sample was scanned five times, and the mean of these values is displayed. For titration analysis, SCCA-1 and SCCA-2 (1 M) in cathepsin assay buffer were titrated with heparin (0.5-10 M), and fluorescence measurements (using an excitation wavelength of 295 nm and an emission wavelength of 340 nm) were recorded. The change in fluorescence (⌬F) divided by the initial fluorescence value (F 0 ) was plotted against heparin concentration, and binding constants were estimated using nonlinear regression analysis. Biotinylation of Lysine Residues-Biotin was covalently linked to rSCCA-1 or rSCCA-2 using Sulfo-NHS-Biotin (sulfo-N-hydroxysuccinimide-LC-biotin; Pierce) following the manufacturer's protocol. Briefly, 0.5 mg of recombinant protein in phosphate-buffered saline was added to 250 l of 1 mg/ml Sulfo-NHS-Biotin and incubated for 1 h at room temperature, and the reaction mixture was dialyzed to remove any free biotinylation reagent. Both biotinylated and unmodified proteins were subjected to heparin affinity chromatography as described above, and eluted fractions were analyzed by SDS-PAGE. Determination of the Association Rate Constant (k a ) of Cathepsin L Inhibition in the Presence and Absence of Heparin- The association constants for inhibition of cathepsin L (EC 3.4.22.15) were determined using the discontinuous method at pH 5.5 in cathepsin assay buffer at room temperature with excitation/emission wavelengths of 370/460 nm (Hitachi F4500 fluorometer). For assays in the absence of heparin, the concentration of cathepsin L (Athens Research) was held at 10 nM, and the concentration of rSCCA-1 was at least 3-fold higher, ranging from 30 to 80 nM. In the presence of heparin, the enzyme concentration was lowered to 2.5 nM with rSCCA-1 concentrations of 7.5 nM. The unfractionated heparin concentration of 0.32 g/ml used in these assays represents a concentration of ϳ30 nM (taking an average molecular mass of 11,000) or 4-fold higher than the concentration of serpin. Following the addition of serpin (in the presence or absence of heparin), the residual cathepsin L activity was calculated at various time points in a final volume of 600 l containing 40 M N-benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-AMC) in assay buffer. Substrate cleavage was measured for 2 min, and the natural logarithm of the residual activity was plotted against the time elapsed. Data were analyzed using linear regression analysis in GraphPad Prism software. The slope of this line represents the observed rate of inhibition (Ϫk obs ), and the association rate constant was determined from the slope of a plot of k obs versus inhibitor concentration. For cathepsin G assays, the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Calbiochem) was used, and residual activity was followed at 405 nm. Stoichiometry of Inhibition-The stoichiometry for cathepsin L inhibition by SCCA-1 and SCCA-2 was determined in the absence and presence of heparin. Briefly, 25 nM cathepsin L was incubated with a range of serpin concentrations (5-25 nM) with or without 50 nM heparin. Reactions were incubated for 1 h at 37°C in cathepsin assay buffer, and fractional residual activity was plotted against the serpin/cathepsin L ratio ([I] 0 /[E] 0 ). The stoichiometry of inhibition value was determined as the x intercept value, calculated by linear regression analysis using Prism. Inhibition of Extracellular Cysteine Protease Activity of Cancer Cell Lines-The cancer cell lines MDA-MB-231 (breast carcinoma) and WM793 (melanoma-derived) were grown in 100-mm dishes to 80 -90% confluence, and conditioned media from these cells were collected. Phenylmethylsulfonyl fluoride (1 mM) and EDTA (1 mM) were added, and the cathepsin L-like activity was determined. Briefly, 50 l of conditioned medium was incubated with SCCA-1 (200 nM), heparin (1 mg/ml), or both in combination for 30 min, in addition to a control bufferonly incubation. Subsequently, 50 l of cathepsin assay buffer was added, and samples were assayed for cathepsin L-like activity over 10 -20 min using the fluorogenic substrate Z-FR-AMC (40 M). The sensitivity of activity to E-64 (10 M) and resistance to CA-074 (10 M) were determined to verify that activity was not due to cathepsin B. The control assay was taken as 100% activity. Each assay was performed in triplicate. Serpins SCCA-1 and SCCA-2 Bind the Glycosaminoglycan Heparin-Although many serpins are known to bind and be modulated by glycosaminoglycans, most notably heparin, this has not previously been investigated for SCCA-1 and SCCA-2. As members of the B-clade subfamily, they are generally regarded as intracellular proteins, but along with other members such as PAI-2 and ovalbumin, an extracellular presence is evident despite the lack of a classical secretion signal sequence (23). We initially examined if the recombinant proteins could bind heparin using heparin-agarose affinity chromatography. Although both proteins are acidic overall, with a predicted and determined pI of Ͻ6.5 (20), we found that they bound relatively tightly to heparin-agarose at pH 6.9 (Fig. 1). Elution required 0.3 M NaCl for SCCA-2 and 0.4 -0.5 M NaCl for SCCA-1. In comparison, a similarly tagged and purified recombinant ovalbumin did not bind heparin-agarose, and antithrombin III was eluted at 1 M NaCl. Binding was also investigated using mobility shift analysis on native PAGE gels. As shown in Fig. 1b, both proteins showed a mobility shift toward the positive electrode following incubation with heparin. We also examined the ability to bind DNA using agarose gel mobility shift analysis but found no binding for either serpin (data not shown), in agreement with the findings of Ong et al. (21) for SCCA-1. Glycosaminoglycan Specificity-To determine the specificity of SCCA-1 and SCCA-2 for various glycosaminoglycans, we used recombinant serpin immobilized by pulldown on heparinagarose beads. The ability of heparin, heparan sulfate, chondroitin sulfates A and B, acetyl heparin, and de-N-sulfated heparin to elute the protein was examined (Fig. 2a). Heparin and heparan sulfate eluted the protein, but as expected, the modified heparin molecules acetyl heparin and de-N-sulfated heparin were unable to elute rSCCA-1 or rSCCA-2, as they lack the negatively charged sulfide groups that mediate the ionic interaction. However, we also noted that chondroitin sulfates A and B were similarly unable to elute rSCCA-1 or rSCCA-2 even at a 10-fold higher concentration than that required for heparin and heparan sulfate, indicating a high degree of specificity for heparin. Further evidence for an ionic interaction was obtained from modification of lysine residues by biotinylation (Fig. 2b). This significantly reduced binding of SCCA-1 and SCCA-2 to heparin-agarose, indicating that surface lysines are important for the interaction and consistent with the lack of binding of desulfated heparin seen in Fig. 2a. Binding of Heparin Quenches the Intrinsic Tryptophan Fluorescence of SCCA-1 and SCCA-2-Cofactor-induced conformational change is a common occurrence in serpin activity, and we examined this by monitoring the change in intrinsic fluorescence. SCCA-1 and SCCA-2 contain 4 and 5 tryptophan residues, respectively (Trp 150 , Trp 186 , Trp 201 , and Trp 269 , with Trp 319 in SCCA-2 alone). The tryptophan fluorescence spectra showed a substantial shift in the presence of 1-5 M heparin for both proteins. A titration curve of change in fluorescence against heparin concentration yielded dissociation constants of 4.20 Ϯ 0.46 M for SCCA-1 and 2.03 Ϯ 0.15 M for SCCA-2 (Fig. 3). Heparin Accelerates the Inhibition of Cathepsin L by SCCA-1 and SCCA-2-Heparin is known to enhance the inhibitory activity of plasma serpins toward their target proteases, with antithrombin III activity toward thrombin increased by Ͼ1000fold in the presence of heparin. However, heparin enhancement of a serpin in the context of cysteine protease inhibition has not previously been reported. We initially investigated if heparin had functional relevance on the ability of SCCA-1 to inhibit lysosomal cathepsin L. We found that, in the presence of heparin, this inhibition was remarkably rapid and complete (Fig. 4a) to the extent that second order inhibition rate constants could not be determined and were estimated at Ͼ10 8 M Ϫ1 s Ϫ1 . In the absence of heparin, the k a was determined as 5.4 ϫ 10 5 M Ϫ1 s Ϫ1 , which is in close agreement with a previous estimate of 3.0 ϫ 10 5 M Ϫ1 s Ϫ1 (4). Thus, in the presence of heparin, the rate of inhibition appears to be increased by at least 180-fold. SCCA-2 shows greater specificity toward serine proteases cathepsin G and mast cell chymase, and we examined effects on cathepsin G activity using the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-pnitroanilide. Interestingly, no increase in inhibitory activity was observed in the presence of heparin in this case (Fig. 4b). SCCA-2 could inhibit cathepsin L but less efficiently than SCCA-1. We found that heparin could also enhance this inhibition but with only a 4.1-fold increase in rate of inhibition (Fig. 4c). For both serpins, the stoichiometry of inhibition was closer to 1:1 in the presence of heparin, indicating that less of the protein is partitioned to the substrate pathway (Table 1). Heparin Enhancement Is a Template Effect-The mechanism for heparin enhancement could depend on binding to serpin alone or on a templating effect whereby both protease and inhibitor are bound, effectively increasing the reactant concentration and rate of inhibition. This template mechanism is found for most plasma serpin enhancement by heparin, but for SCCA-1 rate enhancement in the presence of DNA, a non-templating saturation effect was seen as a result of protease binding only (21). Using a range of heparin concentrations (Fig. 5), we found that a saturating effect did not occur and that the curve obtained was consistent with the formation of a ternary complex, i.e. at high heparin concentrations, the protease and serpin are more likely to bind different heparin molecules, thus decreasing the template effect and rate of inhibition. Furthermore, we found that cathepsin L was able to bind heparin-agarose (eluting at ϳ0.4 M NaCl under the same conditions used for serpin analysis) but that the intrinsic fluorescence of cathepsin L was not altered, suggesting that conformational change in the protease is not induced following heparin binding (data not shown). Heparin and SCCA-1 Combine to Inhibit Cancer Cell Linederived Proteolytic Activity-Overexpression of cathepsin L has been identified in many human malignancies, including mela-nomas and colorectal cancers (19,24,25), and there is also previous evidence that SCCA-1 and heparin can independently inhibit metastasis (14). We investigated if SCCA-1 can inhibit secreted cathepsin L-like activity from two invasive human cancer cell lines and if heparin can enhance this inhibition. We found that conditioned media from the breast cancer cell line FIGURE 4. Effects of heparin on inhibition of cathepsins. a, cathepsin (Cat) L initial rates using the substrate Z-FR-AMC with protease alone and following a 15-s incubation with 7. 5 nM SCCA-1 or ϳ30 nM unfractionated heparin (Hep). Incubation with rSCCA-1 and heparin combined resulted in no detectable residual activity. b, effects of heparin on the inhibition of cathepsin G by SCCA-2. Residual cathepsin G activity with the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is shown for protease alone and following incubation with 50 nM heparin, 100 nM rSCCA-2, and 50 nM heparin ϩ 100 nM rSCCA-2. c, determination of k a for cathepsin L inhibition by rSCCA-1 and rSCCA-2 in the presence and absence of heparin. The initial velocity of substrate cleavage by cathepsin L (5 nM) was determined at various concentrations of serpin. The natural logarithm of the residual activity (ln(E)) was plotted against the time point (s), and the data were fitted using linear regression analysis. The slope of these lines represents Ϫk obs , and a replot of k obs versus serpin concentration yielded the second order rate constant k a . The inactivation of cathepsin L by rSCCA-1 in the presence of heparin was too rapid to determine kinetic data. MDA-MB-231 and the melanoma-derived cell line WM793 possessed substantial ability to cleave the cathepsin L substrate over a 20-min incubation. This cleavage could be largely abolished by the addition of the broad-specificity cysteine protease inhibitor E-64. However, the addition of the cathepsin B inhibitor CA-079 reduced activity by just 15%, and this may account for most of the remaining activity following heparin and SCCA-1 treatment. (Fig. 6). The addition of SCCA-1 alone could partially inhibit the activity, but in the presence of heparin, activity was further reduced to ϳ22% of the original. Potential Heparin-binding Regions of SCCA-1-Several serpin family members can bind heparin, including antithrombin, protease nexin I, heparin cofactor II, plasminogen activator inhibitor I, and protein C inhibitor. The ionic interaction involves positively charged residues of serpins, but the region of the serpin binding to heparin is not highly conserved, i.e. for antithrombin and heparin cofactor II, binding largely involves helix D (26), ␣ 1 -antitrypsin binds via helix F (27), and the protein C inhibitor-binding site is associated with helix H (28). Cardin and Weintraub (29) have identified the motifs xBBBxxBx and xBBxBx as heparin-binding sequences, where B ϭ basic and x ϭ non-basic residues. We examined the SCCAs for such motifs and noted an xBBxBx motif at residues 19 -24 (FRKSKE) in helix A. We carried out site-directed mutagenesis of the motif basic residues ( 20 RKSK 23 to AASA) in SCCA-1 using the QuikChange mutagenesis method (Stratagene). However, the resulting mutant recombinant protein displayed similar affinity for heparin binding and a similar degree of heparin enhancement toward cathepsin L inhibition compared with the wild-type protein (data not shown). The x-ray crystal structure of SCCA-1 has recently been determined (Protein Data Bank code 2ZV6) (30). One exposed residue is the helix D Lys 87 , which is equivalent to Lys 125 in antithrombin III, previously shown by Schedin-Weiss et al. (31) to be important for heparin binding and activation. DISCUSSION The anticancer properties of heparin have been known for many years, but this has not translated to the clinic, and the underlying mechanism is still a subject of some debate (32). Experiments with modified heparins lacking anticoagulant activity suggest that other factors are important, and among those proposed are prevention of platelet interactions with cancer cells by P-selectin binding (33) and competition with cellsurface heparan sulfate proteoglycans for binding proangiogenic growth factors such as fibroblast growth factor-␤ (34). A comprehensive review of the preclinical data has led Niers et al. (35) to conclude that inhibition of metastasis rather than primary tumor growth is the predominant means by which heparin exerts its anticancer effects. Evidence for prometastatic activity of heparanase also supports this hypothesis (36). In acting as an anticoagulant, heparin binds and induces a conformational change in the serpin antithrombin III, greatly accelerating its ability to inhibit thrombin and other coagulation factors via a ternary complex, for which the structure has now been solved (37). Heparin also enhances heparin cofactor II FIGURE 5. Effect of heparin concentration on cathepsin L inhibition by SCCA-1. Cathepsin L residual activity was assessed in the presence of varying heparin concentrations. The optimum heparin concentration range was observed from 2 to 600 nM. The decreased inhibition at higher heparin concentrations indicates a templating mechanism. and protease nexin I inhibition of thrombin and protein C inhibitor inhibition of activated protein C (38,39). A number of serpins can also inhibit angiogenesis, and in the case of latent antithrombin and kallistatin, heparin is found to be important for antiangiogenic activity (40,41), which could represent an indirect mechanism for inhibiting metastasis. However, a more rapid and direct role for heparin in the inhibition of metastatic proteases has not previously been suggested, and our data now underpin this novel mechanism in relation to cathepsin L inhibition. Cathepsin L is a widely expressed cysteine protease with a major role in lysosomal proteolysis, protein processing, matrix degradation, and tissue remodeling, and it has been linked to the invasive phenotype in many cancers (24,25). Inhibition of cathepsin L activity by synthetic inhibitors and by the cathepsin S propeptide can reduce invasiveness of a number of human cancer cell lines (42), and prevention of cathepsin L secretion by introducing an overexpressed intracellular anticathepsin single chain variable antibody fragment dramatically reduced melanoma cell metastasis (43). We propose that SCCA-1 and heparin may combine to facilitate an endogenous mechanism for regulation of extracellular cathepsin activity and cathepsin-mediated metastasis. SCCA-1 is expressed in many epithelial tissues prone to carcinoma development, including skin, cervix, lung, and esophagus. It appears to be predominantly intracellular in normal epithelial cells but is secreted in certain malignancies, in benign disorders such as psoriasis, and following stimulation with specific cytokines. Interestingly, intracellular cathepsin L appears to have a role in processing of proheparanase to the active heparanase (44), facilitating a possible synergistic regulatory role for intracellular SCCA-1. Elucidation of physiologically relevant target proteases for individual serpins can be difficult, and in general, a k a of Ͼ10 3 is considered potentially relevant in vivo. The increase to Ͼ10 8 in the presence of heparin generates an extremely potent and rapid mechanism for cathepsin L inhibition by SCCA-1. To our knowledge, this degree of heparin enhancement (Ͼ180-fold) for cathepsin L activity is the second most significant after thrombin with regard to the protease targeted. -Fold increases with heparin range from 45 to Ͼ2000 for thrombin inhibition by various serpins, but for other proteases, reports range from 4-fold for inhibition of factor XIa to 52-fold for inhibition of activated protein C (28). SCCA-2 is a substantially poorer inhibitor of cathepsin L both alone and in the presence of heparin; we found just a 4-fold heparin-induced increase. Another B-clade serpin, headpin or hurpin (serpin B13), can also inhibit cathepsin L (45), and it remains to be seen if heparin enhances this activity. The inhibition of parasitic and allergen proteases may be a primary function of SCCA-1 and SCCA-2 (8,9), and interestingly, heparin has also been found to have regulatory potential in allergic inflammation (46). Heparin treatment has not shown antimetastatic effects in all preclinical and clinical studies, but successful outcomes do include malignancies where cathepsin L is overexpressed (e.g. B16 melanomas) and tissues in which SCCA-1 has been detected extracellularly (31). A re-examination of heparin preclinical data in terms of serpin and cathepsin expression, secretion, and activity may prove valuable in predicting which cancers will respond positively to heparin treatment.	2018-04-03T01:47:55.442Z	2009-12-03T00:00:00.000	{ "year": 2009, "sha1": "f8da5c30919f9ab87aa4bcb72e8341c68e1f1508", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/285/6/3722.full.pdf", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "fe13e0bfd10cbb0e535761b390738aaefdc1fe4d", "s2fieldsofstudy": [ "Chemistry", "Medicine" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
237280801	pes2o/s2orc	v3-fos-license	"Technological Answerability and the Severance Problem: Staying Connected by Demanding Answers\n\nAr(...TRUNCATED)	2021-08-25T06:16:43.831Z	2021-08-24T00:00:00.000	{"year":2021,"sha1":"58f4ea593f42b6d6511d28161cd98bc191e5d3cc","oa_license":"CCBY","oa_url":"https:/(...TRUNCATED)

End of preview. Expand in Data Studio

YAML Metadata Warning: empty or missing yaml metadata in repo card (https://huggingface.co/docs/hub/datasets-cards)

The arXiv data used in LLMs Know More About Numbers than They Can Say (arXiv:2602.07812, code).

The dataset is derived from allenai/peS2o.

Downloads last month: 8

Size of downloaded dataset files:

2.88 GB

Size of the auto-converted Parquet files:

1.42 GB

Number of rows:

100,000

Paper for VCY019/Numeracy-Probing

LLMs Know More About Numbers than They Can Say

Paper • 2602.07812 • Published 4 days ago